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Several antimicrobial combination therapies are used to treat multiple drug resistant (MDR) and extensively drug resistant (XDR) Acinetobacter baumannii infections. A novel antibiotic, eravacycline, shows a higher potency than tigecycline. The efficacies of eravacycline-based therapies have not yet been evaluated. We demonstrated the effectiveness of eravacycline- and tigecycline-based combination therapies in XDR and especially tigecycline resistant A. baumannii. Thirteen eligible isolates were selected from 642 non-duplicate Acinetobacter blood isolates from four medical centres in 2010-2014. Tigecycline/imipenem and eravacycline/imipenem combinations were simultaneously effective against some isolates in vitro with fractional inhibitory concentration index of 0.5. In contrast, eravacycline- and tigecycline-based combination therapies provided no additional benefits in mouse survival compared to those for monotherapy. In summary, colistin is still the final resort for XDR-A. baumannii treatment according to the sensitivities. Owning to rapid development of resistance in A. baumannii, novel antibiotics are urgently needed.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

ObjectivesTo investigate the susceptibility of imipenem-non-susceptible Escherichia coli (INS-EC), Klebsiella pneumoniae (INS-KP), Acinetobacter baumannii (INS-AB) and Pseudomonas aeruginosa (INS-PA) to novel antibiotics.MethodsMICs were determined using the broth microdilution method. Carbapenemase and ESBL phenotypic testing and PCR for genes encoding ESBLs, AmpCs and carbapenemases were performed.ResultsZidebactam, avibactam and relebactam increased the respective susceptibility rates to cefepime, ceftazidime and imipenem of 17 INS-EC by 58.8%, 58.8% and 70.6%, of 163 INS-KP by 77.9%, 88.3% and 76.1% and of 81 INS-PA by 45.7%, 38.3% and 85.2%, respectively. Vaborbactam increased the meropenem susceptibility of INS-EC by 41.2% and of INS-KP by 54%. Combinations of β-lactams and novel β-lactamase inhibitors or β-lactam enhancers (BLI-BLE) were inactive against 136 INS-AB. In 58 INS-EC and INS-KP with exclusively blaKPC-like genes, zidebactam, avibactam, relebactam and vaborbactam increased the susceptibility of the partner β-lactams by 100%, 96.6%, 84.5% and 75.9%, respectively. In the presence of avibactam, ceftazidime was active in an additional 85% of 20 INS-EC and INS-KP with exclusively blaOXA-48-like genes while with zidebactam, cefepime was active in an additional 75%. INS-EC and INS-KP with MBL genes were susceptible only to cefepime/zidebactam. The β-lactam/BLI-BLE combinations were active against INS-EC and INS-KP without detectable carbapenemases. For INS-EC, INS-KP and INS-AB, tigecycline was more active than omadacycline and eravacycline but eravacycline had a lower MIC distribution. Lascufloxacin and delafloxacin were active in Conclusionsβ-Lactam/BLI-BLE combinations were active in a higher proportion of INS-EC, INS-KP and INS-PA. The susceptibility of novel fluoroquinolones and tetracyclines was not superior to that of old ones.

Objectives We aimed to determine susceptibilities of Elizabethkingia spp. to 25 commonly tested and 8 novel antibiotics, and to compare the performance of different susceptibility testing methods. Methods Clinical isolates of Elizabethkingia spp., Chryseobacterium spp. and Flavobacterium spp. collected during 2002–18 (n = 210) in a nationwide surveillance programme in Taiwan were speciated by 16S rRNA sequencing. MICs were determined by broth microdilution. The broth microdilution results of 18 common antibiotics were compared with those obtained by the VITEK 2 automated system. Results Among the Elizabethkingia spp. identified (n = 108), Elizabethkingia anophelis was the most prevalent (n = 90), followed by Elizabethkingia meningoseptica (n = 7) and Elizabethkingia miricola cluster [E. miricola (n = 6), Elizabethkingia bruuniana (n = 3) and Elizabethkingia ursingii (n = 2)]. Most isolates were recovered from respiratory or blood specimens from hospitalized, elderly patients. PFGE showed two major and several minor E. anophelis clones. All isolates were resistant to nearly all the tested β-lactams. Doxycycline, minocycline and trimethoprim/sulfamethoxazole inhibited >90% of Elizabethkingia spp. Rifampin inhibited E. meningoseptica (100%) and E. anophelis (81.1%). Fluoroquinolones and tigecycline were active against E. meningoseptica and E. miricola cluster isolates. Novel antibiotics, including imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, delafloxacin, eravacycline and omadacycline were ineffective but lascufloxacin inhibited half of Elizabethkingia spp. The very major discrepancy rates of VITEK 2 were >1.5% for ciprofloxacin, moxifloxacin and vancomycin. Major discrepancy rates were >3% for amikacin, tigecycline, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. Conclusions MDR, absence of standard interpretation criteria and poor intermethod concordance necessitate working guidelines to facilitate future research of emerging Elizabethkingia spp.

Objectives Acinetobacter seifertii, a new member of the Acinetobacter baumannii group, has emerged as a cause of severe infections in humans. We investigated the clinical and molecular characteristics of A. seifertii. Patients and methods This retrospective study enrolled 80 adults with A. seifertii bloodstream infection (BSI) at four medical centres over an 8 year period. Species identification was confirmed by MALDI-TOF MS, rpoB sequencing and WGS. Molecular typing was performed by MLST. Clinical information, antimicrobial susceptibility and the mechanisms of carbapenem and colistin resistance were analysed. Transmissibility of the carbapenem-resistance determinants was examined by conjugation experiments. Results The main source of A. seifertii BSI was the respiratory tract (46.3%). The 28 day and in-hospital mortality rates of A. seifertii BSI were 18.8% and 30.0%, respectively. High APACHE II scores and immunosuppressant therapy were independent risk factors for 28 day mortality. The most common MLST type was ST553 (58.8%). Most A. seifertii isolates were susceptible to levofloxacin (86.2%), and only 37.5% were susceptible to colistin. Carbapenem resistance was observed in 16.3% of isolates, mostly caused by the plasmid-borne ISAba1-blaOXA-51-like genetic structure. A. seifertii could transfer various carbapenem-resistance determinants to A. baumannii, Acinetobacter nosocomialis and other A. seifertii isolates. Variations of pmrCAB and lpxCAD genes were not associated with colistin resistance of A. seifertii. Conclusions Levofloxacin and carbapenems, but not colistin, have the potential to be the drug of choice for A. seifertii infections. A. seifertii can transfer carbapenem-resistance determinants to other species of the A. baumannii group and warrants close monitoring.

Background The impact of appropriate antimicrobial therapy for A. baumannii bacteremic pneumonia has not been well established due to the inclusion of the three phenotypically indistinguishable Acinetobacter species and confounding factors including underlying diseases and severity of infection. This retrospective study aimed to evaluate the impact of appropriate antimicrobial therapy on 14-day mortality in A. baumannii bacteremic pneumonia patients after adjusting for risk factors. Methods This study was conducted at five medical centers in Taiwan between July 2012 and June 2016. A. baumannii species identification was performed using reference molecular methods. Risk factors for 14-day mortality were analyzed via logistic regression. The interaction between the Acute Physiology and Chronic Health Evaluation (APACHE) II score and appropriate antimicrobial therapy was assessed using the logistic model. Results A total of 336 patients with monomicrobial A. baumannii bacteremic pneumonia were included in this study. The overall 14-day mortality rate was 47.3%. The crude mortality of appropriate antimicrobial therapy was 35.9% (57 of 151 patients). Appropriate antimicrobial therapy was associated with a lower mortality after multivariate adjustment (odds ratio [OR], 0.57; 95% confidence interval [CI], 0.34–0.97; p = 0.04), and the effect was influenced by APACHE II score (OR for interaction term, 0.0098; 95% CI, 0.0005–0.1885; p = 0.002). Further analysis demonstrated that appropriate antimicrobial therapy significantly reduced 14-day mortality among the patients with an APACHE II score > 35 (OR 0.0098; 95% CI 0.0005–0.1885). Conclusion Appropriate antimicrobial therapy decreases 14-day mortality of the most severely ill patients with A. baumannii bacteremic pneumonia.

Patients with Acinetobacter baumannii bacteremia treated with antipseudomonal cephalosporins showed higher 14-day mortality than patients treated with antipseudomonal carbapenems. We hypothesized that the bacterial membrane vesicles (BMVs) induced by antipseudomonal cephalosporins are more virulent than BMVs induced by antipseudomonal carbapenems. To simulate the clinical condition with inadequate antimicrobial treatment, carbapenem-resistant A. baumannii was treated with ceftazidime (an antipseudomonal cephalosporin) or imipenem (an antipseudomonal carbapenem) at 1/2 the minimum inhibitory concentration. BMVs and BMV-carried lipopolysaccharide were measured by nanoparticle tracking analysis and western blotting, respectively. Cytokine expression in RAW264.7 macrophages or mice serum induced by the BMVs was determined by ELISA, fluorescent bead-based immunoassay or western blotting. The virulence of the BMVs was assessed in mice. Liquid chromatography tandem-mass spectrometry was used to determine the protein contents of the BMVs. We found that ceftazidime induced a higher number of BMVs (CAZ-BMV), which carried more LPS, and induced higher expression levels of iNOS, IL-1β, and IL-6 in macrophages, higher expression of many cytokines in mice, more neutrophil infiltration in lung interstitium, and higher mortality in mice than imipenem-induced BMVs (IMP-BMV). When adjusted to same amount of LPS, CAZ-BMV still led to higher mortality than IMP-BMV. Proteomic analysis revealed different protein contents in CAZ-BMV and IMP-BMV. In conclusion, A. baumannii BMVs induced by ceftazidime are more virulent than BMVs induced by imipenem.

Background: Clinical characteristics and risk factors for mortality of Acinetobacter bacteremia in cirrhotic patients have not been investigated. Methods: Acinetobacter bacteremia cases from four medical centers were collected from 2009 to 2014, to compare between patients with and without liver cirrhosis. Risk factors for mortality of Acinetobacter bacteremia among cirrhotic patients were identified using multivariate logistic regression. Results: Among the patients with Acinetobacter bacteremia, 72 had liver cirrhosis and 816 had not. Patients with cirrhosis were younger (57.5 [50–71] vs. 72 [50.25–71], p

Acinetobacter baumannii, a Gram-negative bacterium, is an important nosocomial pathogen. Colistin-resistant A. baumannii is becoming a new concern, since colistin is one of the last-line antibiotics for infections by carbapenem-resistant A. baumannii. From 452 carbapenem-resistant isolates collected in a teaching hospital in Taipei, Taiwan, we identified seven that were resistant to colistin. Carbapenem resistance in these isolates is attributed to the presence of carbapenemase gene blaOXA-23 in their genomes. Colistin resistance is presumably conferred by mutations in the sensor kinase domain of PmrB found in these isolates, which are known to result in modification of colistin target lipid A via the PmrB–PmrA–PmrC signal transduction pathway. Overexpression of pmrC, eptA, and naxD was observed in all seven isolates. Colistin resistance mediated by pmrB mutations has never been reported in Taiwan. One of the seven isolates contained three mutations in lpxD and exhibited an altered lipopolysaccharide profile, which may contribute to its colistin resistance. No significant difference in growth rates was observed between the isolates and the reference strain, suggesting no fitness cost of colistin resistance. Biofilm formation abilities of the isolates were lower than that of the reference. Interestingly, one of the isolates was heteroresistant to colistin. Four of the isolates were significantly more virulent to wax moth larvae than the reference.

Background: Acinetobacter baumannii is an important nosocomial pathogen worldwide. Its role in community-acquired infection remains controversial and has rarely been reported. Methods: Patients with monobacterial bloodstream infections caused by genomic species identified A. baumannii, admitted to Taipei Veterans General Hospital between 1999 and 2010, were selected as cases. Controls were defined as patients acquiring infection in a healthcare setting and were matched for age and sex. The clinical, epidemiologic, and microbiological characteristics of cases and controls were compared. Results: Cases presented with shock more frequently and had higher APACHE II scores (25 vs 19, p = 0.005). No significant differences between the two groups were noted in the sources of bloodstream infection and underlying diseases. Multidrug resistance rates were higher in nosocomial A. baumannii isolates then in those acquired in the community (81.5% vs 38.9%, p = 0.002). Patients infected in the community were more likely to receive appropriate antimicrobial therapy than those with hospital-acquired A. baumannii (10/18; 55.6% vs 11/54; 20.4%, p = 0.011). Acquisition in the community (odds ratio [OR] 5.716, 95% confidence interval [CI] 1.021–32.003, p = 0.047), respiratory tract as the infection source (OR 9.514, 95% CI 2.370–38.189, p = 0.001), and immunosuppressive therapy (OR 4.331, 95% CI 1.052–17.832, p = 0.042) were independently associated with increased 14-day mortality among patients with A. baumannii bacteremia in this cohort. Conclusion: Community-acquired bacteremia caused by A. baumannii was rare but associated with a severe outcome. Further investigation of potential virulence factors of community-acquired A. baumannii is required. Keywords: Acinetobacter baumannii, Community-acquired infection, Healthcare-associated infection, Bloodstream infection

Background Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. Methods This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. Results We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89–97%) and specificity (81%; 95% CI, 73–88%) for predicting imipenem non-susceptibility. Conclusion Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.

Background According to our preliminary study, BLI-489 has the potential to inhibit the hydrolysing activity of OXA-51-like β-lactamase produced by carbapenem-resistant Acinetobacter baumannii (CRAb). Objectives In the present study, the in vitro and in vivo activities of imipenem combined with BLI-489 against CRAb producing carbapenem-hydrolysing class D β-lactamases (CHDLs), namely OXA-23, OXA-24, OXA-51 and OXA-58, were determined. Methods A chequerboard analysis of imipenem and BLI-489 was performed using 57 and 7 clinical CRAb isolates producing different CHDLs and MBLs, respectively. Four representative strains harbouring different CHDL genes were subjected to a time–kill assay to evaluate the synergistic effects. An in silico docking analysis was conducted to simulate the interactions between BLI-489 and the different families of CHDLs. The in vivo activities of this combination were assessed using a Caenorhabditis elegans survival assay and a mouse pneumonia model. Results Chequerboard analysis showed that imipenem and BLI-489 had a synergistic effect on 14.3, 92.9, 100, 16.7 and 100% of MBL-, OXA-23-, OXA-24-like-, OXA-51-like- and OXA-58-producing CRAb isolates, respectively. In the time–kill assay, imipenem and BLI-489 showed synergy against OXA-24-like-, OXA-51-like- and OXA-58-, but not OXA-23-producing CRAb isolates after 24 h. The in silico docking analysis showed that BLI-489 could bind to the active sites of OXA-24 and OXA-58 to confer strong inhibition activity. The combination of imipenem and BLI-489 exhibited synergistic effects for the rescue of CRAb-infected C. elegans and mice. Conclusions Imipenem combined with BLI-489 has synergistic effects against CHDL-producing CRAb isolates.

Antimicrobial-resistant (AMR) bacterial infections, including those caused by Acinetobacter baumannii, have emerged as a clinical crisis worldwide. Immunization with AMR determinants has been suggested as a novel approach to combat AMR bacteria, but has not been validated. The present study targeted tigecycline (TGC) resistance determinants in A. baumannii to test the feasibility of this approach. Using bioinformatic tools, four candidates, AdeA, AdeI, AdeK, and TolC, belonging to the resistance-nodulation-division (RND) efflux pump were identified as highly conserved and exposed antigens from 15 A. baumannii genomes. Antisera generated from recombinant proteins showed the capability to reserve Hoechst 33342, a substrate of the efflux pump, in bacterial cells. The rTolC antisera had the highest complement-dependent killing and opsonophagocytosis effect compared to the sera from phosphate-buffered saline immunized mice. Among the antisera, anti-rAdeK-specific antisera decreased the minimal inhibitory concentration of TGC in 26.7% of the tested isolates. Immunization with rAdeK significantly potentiated TGC efficacy in treating TGC-resistant A. baumannii pneumonia in the murine model. The bacterial load (7.5 × 105 vs. 3.8 × 107, p < 0.01) and neutrophil infiltration in the peri-bronchial vasculature region of immunized mice was significantly lower compared to the PBS-immunized mice when TGC was administrated concomitantly. Collectively, these results suggest that active immunization against resistance determinants might be a feasible approach to combat multidrug-resistant pathogens in high risk population.

This retrospective observational study assessed the differences between monomicrobial and polymicrobial A. baumannii bacteremia and identified possible independent risk factors for 14-day mortality. There were 379 patients with A. baumannii bacteremia admitted to a tertiary care center in northern Taiwan between August 2008 and July 2015 enrolled for data analysis. Among them, 89 patients (23.5%) had polymicrobial bacteremia and 290 patients (76.5%) had monomicrobial bacteremia. No significant difference in 14-day mortality was observed between patients with monomicrobial and polymicrobial A. baumannii bacteremia (26.9% vs. 29.2%, p = 0.77). Logistic regression controlled for confounders demonstrated that polymicrobial bacteremia was not an independent predictor of mortality, whereas appropriate antimicrobial therapy was independently associated with reduced mortality. Higher 14-day mortality rates were observed in the polymicrobial bacteremic patients with concomitant isolation of Escherichia coli, Pseudomonas aeruginosa, and Enterobacter spp. from the bloodstream. Compared with patients with monomicrobial multidrug-resistant A. baumannii (MDRAb) bacteremia, those with MDRAb concomitant with Gram-negative bacilli bacteremia had a worse outcome. Polymicrobial A. baumannii bacteremia was not associated with a higher 14-day mortality rate than that of monomicrobial A. baumannii bacteremia, although more deaths were observed when certain Gram-negative bacteria were concomitantly isolated. Appropriate antimicrobial therapy remains an important life-saving measure for A. baumannii bacteremic patients.

Background: The Beijing lineage of Mycobacterium tuberculosis (MTB) is the most predominant MTB strain in Asian countries and is spreading worldwide, however, the East AfricanâIndian (EAI) lineage is also particularly prevalent in many tropical Asian countries. The evolutionary relationships among MTB EAI isolates from Taiwan and those of tropical Asian countries remain unknown. Methods: The EAI strains collected from patients in Taiwan were analyzed using spacer oligonucleotide typing and mycobacterial interspersed repetitive unitâvariable number of tandem repeats (MIRU-VNTR) typing, and compared with published profiles from Cambodia and Singapore to investigate potential epidemiological linkages. Results: Among the three countries, the EAI lineage was most prevalent in Cambodia (60%; Singapore, 25.62%; and Taiwan, 21.85%), having also the highest rates of multidrug resistance and lowest rates of clustering of MTB isolates. We describe a convenient method using seven selected MIRU-VNTR loci for first-line typing to discriminate Beijing and EAI lineages. A potential epidemiological linkage in these tropical Asian countries is also discussed based on a minimum-spanning tree constructed using 24 MIRU-VNTR loci of MTB EAI strains. Conclusion: This study identified evolutionary relationships among MTB EAI isolates from Taiwan and those of two other tropical Asian countries, Cambodia and Singapore. Keywords: East AfricanâIndian family, Mycobacterium tuberculosis, tropical Asian countries

Antibiotic-resistant Acinetobacter baumannii is associated with nosocomial infections worldwide. Here, we used clinically isolated A. baumannii strains as models to demonstrate whether antibiotic resistance is correlated with an increased susceptibility to bacteriophages. In this study, 24 active phages capable of infecting A. baumannii were isolated from various environments, and the susceptibilities of both antibiotic-sensitive and antibiotic-resistant strains of A. baumannii to different phages were compared. In our study, a total of 403 clinically isolated A. baumannii strains were identified. On average, the phage infection percentage of the antibiotic-resistant A. baumannii strains was 84% (from 81–86%), whereas the infection percentage in the antibiotic-sensitive A. baumannii strains was only 56.5% (from 49–64%). In addition, the risk of phage infection for A. baumannii was significantly increased in the strains that were resistant to at least four antibiotics and exhibited a dose-dependent response (p-trend A. baumannii isolates, 75.6% were phage typeable. The results of phage typing might also reveal the antibiotic-resistant profiles of clinical A. baumannii strains. In conclusion, phage susceptibility represents an evolutionary trade-off in A. baumannii strains that show adaptations for antibiotic resistance, particularly in medical environments that have high antibiotic use.

OBJECTIVERalstonia pickettii has caused contamination of pharmaceutical solutions in many countries, resulting in healthcare infections or outbreak events. We determined the source of the outbreak of R. pickettii bloodstream infection (BSI).METHODSThis study was conducted in a 3,000-bed tertiary referral medical center in Taiwan with >8,500 admissions during May 2015. Patients had been treated in the injection room or chemotherapy room at outpatient departments, emergency department, or hospital wards. All patients who were culture positive for R. pickettii from May 3 to June 11, 2015, were eligible for the study. The aim of the survey was to conduct clinical epidemiological and microbiological investigations to identify possible sources of infection.RESULTSWe collected 57 R. pickettii–positive specimens from 30 case patients. We performed 24 blood cultures; 14 of these revealed >2 specimens and 6 used fluid withdrawn from Port-a-Cath implantable venous access devices. All patients received an injection of 20 mL 0.9% normal saline via catheter flushing. In addition, 2 unopened ampules of normal saline solution (20 mL) were confirmed positive for R. pickettii. The Taiwan Centers for Disease Control and Prevention performed sampling and testing of the same manufactured batch and identified the same strain of R. pickettii. Pulsed-field gel electrophoresis tests revealed that all clinical isolates had similarity of >90%, validating the outbreak of the same clone of R. pickettii.CONCLUSIONSR. pickettii can grow in saline solutions and cause bloodstream infections. Hospital monitoring mechanisms are extremely important measures in identifying and ending such outbreaks.Infect Control Hosp Epidemiol 2017;38:444–448

A strong interaction between colistin, a last‐resort antibiotic of the polymyxin family, and free lipopolysaccharide (LPS, also referred to as endotoxin), released from the Gram‐negative bacterial (GNB) outer membrane (OM), has been identified that can decrease the antibacterial efficacy of colistin, potentially increasing the dose of this antibiotic required for treatment. The competition between LPS in the GNB OM and free LPS for the interaction with colistin was prevented by using a supramolecular trap to capture free LPS. The supramolecular trap, fabricated from a subnanometer gold nanosheet with methyl motifs (SAuM), blocks lipid A, preventing the interaction between lipid A and colistin. This can minimize endotoxemia and maximize the antibacterial efficacy of colistin, enabling colistin to be used at lower doses. Thus, the potential crisis of colistin resistance could be avoided., Caught in a trap: The antibiotic colistin targets lipopolysaccharide (LPS) in the Gram‐negative bacterial (GNB) membrane. This interaction is disrupted by free LPS released during infection (path a). A methylated gold nanosheet (SAuM) binds to free LPS, preventing free LPS from binding colistin and reducing endotoxemia (path b). This increases the antibacterial efficacy of colistin, decreasing both the required dose and the risk of resistance.

This study investigated the molecular epidemiology of carbapenem-resistant Acinetobacter nosocomialis and Acinetobacter pittii (ANAP). Clinical isolates of Acinetobacter spp., This study investigated the molecular epidemiology of carbapenem-resistant Acinetobacter nosocomialis and Acinetobacter pittii (ANAP). Clinical isolates of Acinetobacter spp. collected by the biennial nationwide Taiwan Surveillance of Antimicrobial Resistance program from 2010 to 2014 were subjected to species identification, antimicrobial susceptibility testing, and PCR for detection of carbapenemase genes. Whole-genome sequencing or PCR mapping was performed to study the genetic surroundings of the carbapenemase genes. Among 1,041 Acinetobacter isolates, the proportion of ANAP increased from 11% in 2010 to 22% in 2014. The rate of carbapenem resistance in these isolates increased from 7.5% (3/40) to 22% (14/64), with a concomitant increase in their resistance to other antibiotics. The blaOXA-72 and blaOXA-58 genes were highly prevalent in carbapenem-resistant ANAP. Various genetic structures were found upstream of blaOXA-58 in different plasmids. Among the plasmids found to contain blaOXA-72 flanked by XerC/XerD, pAB-NCGM253-like was identified in 8 of 10 isolates. Conjugations of plasmids carrying blaOXA-72 or blaOXA-58 to A. baumannii were successful. In addition, three isolates with chromosome-located blaOXA-23 embedded in AbGRI1-type structure with disruption of genes other than comM were detected. Two highly similar plasmids carrying class I integron containing blaIMP-1 and aminoglycoside resistance genes were also found. The universal presence of blaOXA-272/213-like on A. pittii chromosomes and their lack of contribution to carbapenem resistance indicate its potential to be a marker for species identification. The increase of ANAP, along with their diverse mechanisms of carbapenem resistance, may herald their further spread and warrants close monitoring.

Acinetobacter baumannii biofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptible A. baumannii (CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms. A. baumannii ATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone.

Minocycline-based combination therapy has been suggested to be a possible choice for the treatment of infections caused by minocycline-susceptible Acinetobacter baumannii , but its use for the treatment of infections caused by minocycline-resistant A. baumannii is not well established. In this study, we compared the efficacy of minocycline-based combination therapy (with colistin, cefoperazone-sulbactam, or meropenem) to that of colistin in combination with meropenem for the treatment of minocycline-resistant A. baumannii infection. From 2006 to 2010, 191 (17.6%) of 1,083 A. baumannii complex isolates not susceptible to minocycline from the Taiwan Surveillance of Antimicrobial Resistance program were collected. Four representative A. baumannii isolates resistant to minocycline, amikacin, ampicillin-sulbactam, ceftazidime, ciprofloxacin, cefepime, gentamicin, imipenem, levofloxacin, meropenem, and piperacillin-tazobactam were selected on the basis of the diversity of their pulsotypes, collection years, health care setting origins, and geographic areas of origination. All four isolates had tetB and overexpressed adeABC , as revealed by quantitative reverse transcription-PCR. Among all minocycline-based regimens, only the combination with colistin produced a fractional inhibitory concentration index comparable to that achieved with meropenem combined with colistin. Minocycline (4 or 16 μg/ml) in combination with colistin (0.5 μg/ml) also synergistically killed minocycline-resistant isolates in time-kill studies. Minocycline (50 mg/kg of body weight) in combination with colistin (10 mg/kg) significantly improved the survival of mice and reduced the number of bacteria present in the lungs of mice compared to the results of monotherapy. However, minocycline (16 μg/ml)-based therapy was not effective at reducing biofilm-associated bacteria at 24 or 48 h when its effectiveness was compared to that of colistin (0.5 μg/ml) and meropenem (8 μg/ml). The clinical use of minocycline in combination with colistin for the treatment of minocycline-resistant A. baumannii may warrant further investigation.

Background Sulbactam is an effective antimicrobial agent against multidrug-resistant Acinetobacter spp. This retrospective study evaluated the risk factors of sulbactam nonsusceptibility (SNS) in monomicrobial Acinetobacter nosocomialis bacteremia and its related outcome. Methods This 9-year retrospective study included 267 patients who were admitted to a large teaching hospital in Taiwan with monomicrobial A. nosocomialis bacteremia. A. nosocomialis was identified to the species level using molecular methods. Antimicrobial susceptibilities were determined by the agar dilution method. To identify the risk factors of acquiring resistant strains, significant clinical variables derived from univariate analysis were entered into multivariate analysis. Polymerase chain reaction was used to identify bla TEM . Clonality was determined by pulsed-field gel electrophoresis. Results A total of 41 of the 267 patients (15.4%) had SNS A. nosocomialis bacteremia. Compared to those with susceptible strains, these patients had higher 14-day mortality (17.1% vs. 7.5%, p = 0.049), were more likely to have higher Acute Physiology and Chronic Health Evaluation (APACHE) II score, were more frequently admitted to the intensive care unit, and had previously received broad-spectrum antibiotics and underwent invasive procedures. In multivariate analysis, the independent risk factors were high APACHE II score and prior use of arterial line [odds ratio (OR), 1.048; 95% confidence interval (CI), 1.007–1.091; p = 0.022 and OR, 2.936; 95% CI, 1.339–6.441; p = 0.007, respectively]. No outbreak was identified and SNS isolates did not harbor bla TEM . Conclusion For monomicrobial A. nosocomialis bacteremia, the mortality of patients with SNS strains was higher. The SNS strains are more commonly recovered from patients with higher APACHE score and receiving more invasive procedures, especially arterial line.

We aimed to establish the role of MALDI-TOF MS on species discrimination of phenotypically indistinguishable A. baumannii, A. pittii and A. nosocomialis. Compared to multiplex PCR, the gold standard, MALDI-TOF MS yielded a high sensitivity for A. baumannii (97.9%) and specificity for A. pittii (98.9%) and A. nosocomialis (100%).

Colistin remains a last-line antibiotic for the treatment of infections by multidrug-resistant Acinetobacter species. However, mortality rates are high in patients with Acinetobacter infection receiving colistin treatment. This multicentre study evaluated whether colistin susceptibility, additional antimicrobial agents or other prognostic factors influenced the clinical outcomes of patients receiving colistin treatment for Acinetobacter bacteraemia. This retrospective study enrolled 122 adults receiving colistin for monomicrobial Acinetobacter bacteraemia at six medical centres in the ACTION Study Group over an 8-year period. Clinical information, antimicrobial susceptibility and colistin resistance determinants were analysed. The primary outcome measure was 14-day mortality. Among 122 patients, 18 and 104 were infected with colistin-resistant (ColR) isolates [minimum inhibitory concentration (MIC) ≥4 mg/L] and colistin-susceptible (ColS) isolates (MIC ≤2 mg/L), respectively. Patients infected with ColR and ColS isolates did not differ significantly with regard to Charlson comorbidity index, invasive procedures, sources of bacteraemia, disease severity and 14-day mortality rate (44.4% vs. 34.6%; P = 0.592). No specific additional antimicrobial agent was independently associated with higher or lower mortality. Coronary artery disease, higher Acute Physiology and Chronic Health Evaluation (APACHE) II score and bacteraemia caused Acinetobacter baumannii were independent risk factors associated with 14-day mortality. Mechanisms of colistin resistance were associated with amino acid variants in the pmrCAB operon. Finally, previously unreported Acinetobacter nosocomialis amino acid variants related to colistin resistance were identified. In conclusion, colistin susceptibility and colistin combination antimicrobial treatment were not associated with decreased 14-day mortality in patients with Acinetobacter bacteraemia receiving colistin treatment.

The aim of this study was to investigate the susceptibility of respiratory Gram-negative bacteria to ceftolozane/tazobactam and other antibiotics in the Asia-Pacific region during 2015-2016. MICs were determined using the CLSI standard broth microdilution method and interpreted accordingly. Pseudomonas aeruginosa (1574 isolates), Klebsiella pneumoniae (1226), Acinetobacter baumannii (627) and Escherichia coli (476) accounted for 73.1% of 5342 Gram-negative respiratory pathogens. Susceptibility to ceftolozane/tazobactam of individual Enterobacteriaceae was >80%, except for Enterobacter cloacae (76.6%). Ceftolozane/tazobactam inhibited 81.9% of K. pneumoniae and 91.9% of E. coli, with respective MIC50/MIC90 values of 0.5/>32 and 0.25/2 mg/L. For carbapenem-susceptible, ESBL-producing K. pneumoniae and E. coli, susceptibility was 65.5% and 93.3%, respectively, and respective MIC50/MIC90 values were 2/>32 and 0.5/2 mg/L. BlaCTX-M-1 group was most prevalent in selected ESBL-producing K. pneumoniae (40 of 54 isolates) and E. coli (15 of 22 isolates), with ceftolozane/tazobactam susceptibility rates of 50% and 80%, respectively. BlaSHV-ESBL was the second most prevalent, and ceftolozane/tazobactam inhibited 20% of 20 K. pneumoniae isolates with blaSHV-ESBL. The only effective antibiotics for carbapenem-non-susceptible K. pneumoniae (111 isolates) and E. coli (24 isolates) were amikacin and colistin. Ceftolozane/tazobactam was effective against almost all tested P. aeruginosa and carbapenem-non-susceptible strains, with susceptibility of 92.3% and 72.8%, respectively; the respective MIC50/MIC90 values were 1/4 and 2/>32 mg/L. The high susceptibility of ceftolozane/tazobactam remained in different age groups, patient locations, recovery times and countries, except Vietnam. In conclusion, ceftolozane/tazobactam was effective against most respiratory Gram-negative pathogens in the Asia-Pacific region; however, the emergence of carbapenem resistance mandates ongoing surveillance.

Amidst the ever-rising threat of antibiotics resistance, colistin, a decade-old antibiotic with lingering toxicity concern, is increasingly prescribed to treat many drug-resistant, gram-negative bacteria. With the aim of improving the safety profile while preserving the antimicrobial activity of colistin, a nanoformulation is herein developed through coacervate complexation with polyanionic peptides. Upon controlled mixing of cationic colistin with polyglutamic acids, formation of liquid coacervates was demonstrated. Subsequent stabilization by DSPE-PEG and homogenization through micro-fluidization of the liquid coacervates yielded nanoparticles 8 nm in diameter. In vitro assessment showed that the colistin antimicrobial activity against multiple drug-resistant bacterial strains was retained and, in some cases, enhanced following the nanoparticle assembly. In vivo administration in mice demonstrated improved safety of the colistin nanoparticle, which has a maximal tolerated dose of 12.5 mg/kg compared to 10 mg/kg of free colistin. Upon administration over a 7-day period, colistin nanoparticles also exhibited reduced hepatotoxicity as compared to free colistin. In mouse models of Klebsiella pneumoniae bacteremia and Acinetobacter baumannii pneumonia, treatment with colistin nanoparticles showed equivalent efficacy to free colistin. These results demonstrate coacervation-induced nanoparticle assembly as a promising approach towards improving colistin treatments against bacterial infections. Statement of Significance Improving the safety of colistin while retaining its antimicrobial activity has been a highly sought-after objective toward enhancing antibacterial treatments. Herein, we demonstrate formation of stabilized colistin nanocomplexes in the presence of anionic polypeptides and DSPE-PEG stabilizer. The nanocomplexes retain colistin’s antimicrobial activity while demonstrating improved safety upon in vivo administration. The supramolecular nanoparticle assembly of colistin presents a unique approach towards designing antimicrobial nanoparticles.

Purpose: To investigate the increasing prevalence of colistin resistance in animal Escherichia coli isolates and their mcr-1-carrying plasmids, especially those shared by isolates from human and retail meats. Methods: E. coli from diseased swine and poultry recovered between 2012 and 2016 were studied. Susceptibility was determined using broth microdilution method or Vitek II system. Fifty-eight mcr-1-positive isolates were randomly selected for further testing, including pulsed-field gel electrophoresis (PFGE) for clonality determination, S1- or I-CeuI-PFGE and Southern blotting for localization of mcr-1, and conjugation for transmissibility. Whole genome sequencing (WGS) was performed for the genetic structure of plasmids. Results: Among the 1234 E. coli isolates from diseased swine, colistin resistance increased from 14.6% (14/96) in 2012 to 43.8% (63/144) in 2016 with a paralleled increase in mcr-1-positivity from 12.5% (12/96) to 33.3% (48/144) in 2016 (P

The emergence of antibiotic-resistant bacteria has threatened our health worldwide. There is an urgent need for novel antibiotics. Previously, we identified a novel 37-mer antimicrobial peptide (AMP), HBcARD, with broad spectrum antimicrobial activity. Here, we improved the efficacy of HBcARD, by re-engineering the peptide, including the addition of a new cysteine to its C-terminus (CTC). The new 28-mer derivative, D-150-177C, contains all D-form arginines, in addition to a C-terminal cycteine. This peptide can kill antibiotic-resistant clinical isolates of Gram-negative bacteria, and is more potent than the parental HBcARD peptide in a mouse sepsis model. In another lung infection mouse model, D-150-177C showed protection efficacy against colistin-resistant Acinetobacter baumannii. Unlike colistin, we observed no acute toxicity of D-150-177C in vivo. Interestingly, we found that CTC modification could enhance the antibacterial activity of several other AMPs, such as buforinII and lysin. The potential application and mechanism of this CTC method as a general approach to improving drug efficacy, warrants further investigation in the future.

Endotoxicity originating from a dangerous debris (i.e., lipopolysaccharide, LPS) of Gram-negative bacteria is a challenging clinical problem, but no drugs or therapeutic strategies that can successfully address this issue have been identified yet. In this study, we report a subnanometer gold cluster that can efficiently block endotoxin activity to protect against sepsis. The endotoxin blocker consists of a gold nanocluster that serves as a flakelike substrate and a coating of short alkyl motifs that act as an adhesive to dock with LPS by compacting the intramolecular hydrocarbon chain-chain distance ( d-spacing) of lipid A, an endotoxicity active site that can cause overwhelming cytokine induction resulting in sepsis progression. Direct evidence showed the d-spacing values of lipid A to be decreased from 4.19 Å to either 3.85 or 3.54 Å, indicating more dense packing densities in the presence of subnanometer gold clusters. In terms of biological relevance, the concentrations of key pro-inflammatory NF-κB-dependent cytokines, including plasma TNF-α, IL-6, and IL-1β, and CXC chemokines, in LPS-challenged mice showed a noticeable decrease. More importantly, we demonstrated that the treatment of antiendotoxin gold nanoclusters significantly prolonged the survival time in LPS-induced septic mice. The ultrasmall gold nanoclusters could target lipid A of LPS to deactivate endotoxicity by compacting its packing density, which might constitute a potential therapeutic strategy for the early prevention of sepsis caused by Gram-negative bacterial infection.

Objectives Tigecycline non-susceptible Acinetobacter nosocomialis (TNAN) has been discovered in clinical isolates. The resistance-nodulation-cell division (RND)-type efflux system plays a major role in tigecycline non-susceptible Acinetobacter baumannii, but the mechanism in A. nosocomialis remains unknown. Our aim was to analyse the contribution of efflux-based tigecycline resistance in clinical A. nosocomialis isolates collected from multiple medical centres in Taiwan. Methods A total of 57 A. nosocomialis isolates, including 46 TNAN and 11 tigecycline-susceptible A. nosocomialis (TSAN) isolates, were analysed. Of these, 46 TNAN isolates were clustered to ST410 (43 isolates) and ST68 (three isolates) by multi-locus sequence typing. Results The relationship between the RND efflux pump and tigecycline resistance was indirectly verified by successfully reducing tigecycline resistance with NMP, an efflux pump inhibitor. The three RND efflux systems (AdeABC, AdeIJK and AdeFGH) were detected in all clinical isolates. The transcript level of adeB gene increased significantly and was correlated with tigecycline resistance. Moreover, the AdeRS two-component system was further classified into four different types of AdeRS patterns considering the amino acid sequence. Further analysis showed that tigecycline resistance was related to the transcript level of adeB gene and the AdeRS pattern. Conclusion This study showed that the dissemination of TNAN isolates in Taiwan is attributable mainly to the spread of ST410. The AdeABC efflux pump appeared to play an important role in the tigecycline resistance of A. nosocomialis.

The rate of recovery of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates has increased significantly in recent decades in Taiwan. This study investigated the molecular epidemiology of CRAB with a focus on the mechanisms of resistance and spread in isolates with bla OXA-23-like or bla OXA-24-like . All 555 CRAB isolates in our multicenter collection, which were recovered from 2002 to 2010, were tested for the presence of class A, B, and D carbapenemase genes. All isolates with bla OXA-23-like or bla OXA-24-like were subjected to pulsed-field gel electrophoresis, and 82 isolates (60 isolates with bla OXA-23-like and 22 isolates with bla OXA-24-like ) were selected for multilocus sequence typing to determine the sequence type (ST) and clonal group (CG) and for detection of additional β-lactamase and aminoglycoside resistance genes. The flanking regions of carbapenem and aminoglycoside resistance genes were identified by PCR mapping and sequencing. The localization of bla OXA was determined by S1 nuclease and I-CeuI assays. The numbers of CRAB isolates carrying bla OXA-23-like or bla OXA-24-like , especially those carrying bla OXA-23-like , increased significantly from 2008 onward. The bla OXA-23-like gene was carried by antibiotic resistance genomic island 1 (AbGRI1)-type structures located on plasmids and/or the chromosome in isolates of different STs (CG92 and novel CG786), whereas bla OXA-24-like was carried on plasmids in CRAB isolates of limited STs (CG92). No class A or B carbapenemase genes were identified. Multiple aminoglycoside resistance genes coexisted in CRAB. Tn 6180 -borne armA was found in 74 (90.2%) CRAB isolates, and 58 (70.7%) isolates had Tn 6179 upstream, constituting AbGRI3. bla TEM was present in 38 (46.3%) of the CRAB isolates tested, with 35 (92.1%) isolates containing bla TEM in AbGRI2-type structures, and 61% of ampC genes had IS Aba1 upstream. We conclude that the dissemination and spread of a few dominant lineages of CRAB containing various resistance island structures occurred in Taiwan.

OBJECTIVES To investigate the link between two NDM-1-positive Acinetobacter isolates from the same hospital, the plasmid profiles of the isolates were examined. These two isolates were found from a surveillance programme within 3 months from two patients without obvious physical contact or hospitalization time overlap. METHODS Antimicrobial susceptibility tests, genome sequencing of both isolates and plasmid transfer experiments were performed. A comparative study of similar plasmids was performed using BLAST analysis. RESULTS The antimicrobial susceptibility of the isolates (Acinetobacter soli M131 and Acinetobacter pittii MS32) and their Escherichia coli transconjugants revealed a conjugative plasmid that carried the carbapenem resistance determinant. Eleven plasmids were observed in M131 and three in MS32. Each isolate shared an identical plasmid that carried the blaNDM-1 gene. This 47 271 bp plasmid harbours a conserved blaNDM-1-containing region that is flanked by ISAba125 and ISAba11 elements, and also contains a Ti-type conjugative operon. The plasmid is nearly identical in sequence to those of Acinetobacter isolates from China. In contrast to the mobilization of the blaNDM-1 sequence in Enterobacteriaceae, which is mainly by transposition, this plasmid moves as a whole among Acinetobacter species. Consistently, this plasmid was found to transfer effectively by in vitro conjugation to several Acinetobacter species. CONCLUSIONS The clinical and laboratory findings suggest that Acinetobacter species may serve as a reservoir of this blaNDM-1 plasmid. Our study demonstrates the potential of applying genome sequencing to the surveillance of antimicrobial-resistant bacteria.

We describe the clinical outcome of 17 patients with secondary Acinetobacter bacteremia whose isolates had a tigecycline MIC of ≤2 mg/liter and who received tigecycline within 2 days of bacteremia onset. The 14-day mortality rate of the tigecycline cohort was 41.2% (7/17), which was significantly higher than that of those receiving other appropriate antimicrobial agents (13.8%, 9/65; P = 0.018). However, the percentages of end-stage renal disease and congestive heart failure were higher in the tigecycline cohort. The efficacy of tigecycline was contingent upon the illness severity and bacterial species. Tigecycline should be applied cautiously for treatment of Acinetobacter bacteremia.

Background Extended-spectrum β-lactamase (ESBL)-producing pathogens have been increasingly identified in community-onset urinary tract infection (UTI). This study was conducted to determine the epidemiology and risk factors of community-onset UTI caused by ESBL-producing pathogens, and to determine the correlation of antimicrobial resistance with ESBL detected by phenotypic and genotypic methods. Methods The study was conducted from December 2010 to January 2012. Patients with community-onset UTI caused by Enterobacteriaceae were enrolled from the emergency department. The production of ESBL was determined by the phenotypic method (using the combined disk test) or by the genotypic method (using polymerase chain reaction detection). The patients' medical records were reviewed and risk factors were analyzed by multivariate analysis. Results A total of 376 patients were enrolled and 393 isolates from urine culture were analyzed. Escherichia coli was the most commonly isolated species (259/393 isolates; 65.9%), followed by Klebsiella pneumoniae (42/393 isolates; 10.7%). Fifty-three (13.5%) isolates were phenotypically positive for ESBL production. Nine (2.3%) isolates were phenotypically positive for both ESBL and AmpC β-lactamase (AmpC) production. Nasogastric tube placement [odds ratio (OR) 2.230; 95% confidence interval (CI) 1.244–3.997; p = 0.007] and hospitalization within the previous 3 months (OR 2.567, 95% CI 1.448–4.551, p = 0.001) were independently associated with the acquisition of ESBL-producing pathogens in community-onset UTI. The ESBL phenotype had a better correlation with resistance to third-generation cephalosporins, compared to the ESBL-positive genotype. Conclusion In our study, nasogastric tube placement and hospitalization within the previous 3 months were significantly associated with the acquisition of ESBL-producing pathogens in community-onset UTI.

Acinetobacter baumannii (Ab) is a global emerging bacterium causing nosocomial infections such as pneumonia, meningitis, bacteremia and soft tissue infections especially in intensive care units. Since Ab is resistant to almost all conventional antibiotics, it is now one of the 6 top-priorities of the dangerous microorganisms listed by the Infectious Disease Society of America. The development of vaccine is one of the most promising and cost-effective strategies to prevent infections. In this study, we identified potential protective vaccine candidates using reverse vaccinology. We have analyzed 14 on-line available Ab genome sequences and found 2752 homologous core genes. Using information obtained from immuno-proteomic experiments, published proteomic information and the bioinformatics PSORTb v3.0 software to predict the location of extracellular and/or outer membrane proteins, 77 genes were identified and selected for further studies. After excluding those antigens have been used as vaccine candidates reported by the in silico search-engines of PubMed and Google Scholar, 13 proteins could potentially be vaccine candidates. We have selected and cloned the genes of 3 antigens that were further expressed and purified. These antigens were found to be highly immunogenic and conferred partial protection (60%) in a pneumonia animal model. The strategy described in the present study incorporates the advantages of reverse vaccinology, bioinformatics and immuno-proteomic platform technologies and is easy to perform to identify novel immunogens for multi-component vaccines development.

BackgroundCarbapenem-resistant Acinetobacter baumannii has emerged as an important cause of nosocomial infections with high morbidity and mortality. The carbapenemases, especially class D carbapenem-hydrolyzing oxacillinases (CHDLs), play an important role, but the relationship between their prevalence trend and carbapenem resistance remains unclear.Materials and methodsBetween 1995 and 2012, we collected 667 isolates of A. baumannii from a single medical center in northern Taiwan. Pulsed-field gel electrophoresis (PFGE) was used to determine clonality. Antimicrobial susceptibility was determined. Carbapenemase genes and associated genetic structures were detected by polymerase chain reaction.ResultsIsolates were heterogeneous on PFGE. Susceptibility to carbapenem decreased steadily over the study period from 88.1% (2001–2003) to

Breakthrough Acinetobacter bacteremia during carbapenem therapy is not uncommon, and it creates therapeutic dilemmas for clinicians. This study was conducted to evaluate the clinical and microbiological characteristics of breakthrough Acinetobacter bacteremia during carbapenem therapy and to assess the efficacy of various antimicrobial therapies. We analyzed 100 adults who developed breakthrough Acinetobacter bacteremia during carbapenem therapy at 4 medical centers over a 6-year period. Their 30-day mortality rate was 57.0%, and the carbapenem resistance rate of their isolates was 87.0%. Among patients with carbapenem-resistant Acinetobacter bacteremia, breakthrough bacteremia during carbapenem therapy was associated with a significantly higher 14-day mortality (51.7% versus 37.4%, respectively; P = 0.025 by bivariate analysis) and a higher 30-day mortality ( P = 0.037 by log rank test of survival analysis) than in the nonbreakthrough group. For the treatment of breakthrough Acinetobacter bacteremia during carbapenem therapy, tigecycline-based therapy was associated with a significantly higher 30-day mortality (80.0%) than those with continued carbapenem therapy (52.5%) and colistin-based therapy (57.9%) by survival analysis ( P = 0.047 and 0.045 by log rank test, respectively). Cox regression controlling for confounders, including severity of illness indices, demonstrated that treatment with tigecycline-based therapy for breakthrough Acinetobacter bacteremia was an independent predictor of 30-day mortality (hazard ratio, 3.659; 95% confidence interval, 1.794 to 7.465; P < 0.001). Patients with breakthrough Acinetobacter bacteremia during carbapenem therapy posed a high mortality rate. Tigecycline should be used cautiously for the treatment of breakthrough Acinetobacter bacteremia that develops during carbapenem therapy.

The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) offer different recommendations for carbapenem MIC susceptibility breakpoints for Acinetobacter species. In addition, the clinical efficacy of the intermediate category remains uncertain. This study was designed to determine the optimal predictive breakpoints based on the survival of patients with Acinetobacter bacteremia treated with a carbapenem. We analyzed the 30-day mortality rates of 224 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia at 4 medical centers over a 5-year period, according to the carbapenem MICs of the initial isolates. The 30-day mortality was about 2-fold greater in patients whose isolates had carbapenem MICs of ≥8 mg/liter than in those with isolates with MICs of ≤4 mg/liter. The differences were significant by bivariate analysis (53.1% [60/113] versus 25.2% [28/111], respectively; P < 0.001) and on survival analysis by the log rank test ( P < 0.001). Classification and regression tree analysis revealed a split between MICs of 4 and 8 mg/liter and predicted the same difference in mortality, with a P value of Acinetobacter bacteremia caused by isolates with carbapenem MICs of ≥8 mg/liter was an independent predictor of 30-day mortality (odds ratio, 4.218; 95% confidence interval, 2.213 to 8.039; P < 0.001). This study revealed that patients with Acinetobacter bacteremia treated with a carbapenem had a more favorable outcome when the carbapenem MICs of their isolates were ≤4 mg/liter than those with MICs of ≥8 mg/liter.

The higher 14-day mortality rate for patients with Acinetobacter bacteremia receiving tigecycline appropriately compared to other appropriate antibiotics (36.4% versus 14.2%, P = 0.028) was due to the poor effect of tigecycline for isolates with a minimum inhibitory concentration of 2 μg/mL (63.6% of 11 versus 14.2% of 127, P = 0.001).

Concerns of Acinetobacter baumannii infection have increased due to the emergence of multi-drug resistance. In the present study, we determined the capsular polysaccharide (CPS) structure of A. baumannii SK44, a clinical isolate from Taiwan, to consist of pentasaccharide repeats. We found that CPS-induced antibody provided 55% protection against challenge in an animal model. The CPS-specific antibody reacted with the surface components of about 62% clinical isolates (342/554 strains) from cross-sectional and longitudinal studies by dot-immunoassay. Pulsed-field gel electrophoresis of positive strains showed the antibody covered different clonalites of A. baumannii clinical isolates. Meanwhile, using the CPS antibody as a probe, we found a number of outer membrane proteins bound to the antibody, including OmpA/motB, TonB-dependent receptor, and Omp38, indicating their association with CPS. These results might lead to the use of the capsular polysaccharide as a vaccine to prevent A. baumannii infection.

Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus–A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S–23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those ‘between 1 and 3’ or ‘close to 13TU’. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species.

Background The emergence of imipenem-nonsusceptible (INS) Acinetobacter baumannii complex has had a great impact on healthcare systems worldwide. Understanding the risk factors related to INS infection is useful for infection control. The risk factors for INS A. baumannii have been well documented; however, the risk factors related to INS Acinetobacter nosocomialis infection lack documentation. The purpose of this study was to identify the risk factors associated with INS A. nosocomialis bacteremia. Methods This retrospective 9-year study included 329 adults with A. nosocomialis bacteremia in a tertiary medical center in Taiwan. Acinetobacter nosocomialis was identified using a multiplex polymerase chain reaction method and sequence analysis of a 16S–23S intergenic spacer. Results Among 329 patients with A. nosocomialis bacteremia, 67 had INS isolates (20.4%). Patients with INS isolates tended to have a more severe form of the diseases [with ICU admission and a higher APACHE (Acute Physiology and Chronic Health Evaluation) II score], specific underlying diseases (associated with chronic lung diseases and end-stage renal diseases, but less commonly alcoholism and chemotherapy), multiple invasive procedures, pneumonia as a primary focus of infection, and prior antimicrobial use (sulbactam, antipseudomonal penicillins, aminoglycosides, and carbapenems). Multivariable analysis showed that ICU admission, chronic lung diseases, arterial line catheterization, total parenteral nutrition, and prior use of carbapenems were independent risk factors; prior use of carbapenems was found to be the most influential (odds ratio 6.36, 95% confidence interval 2.00–20.21; p = 0.002). Conclusion To our knowledge, this is the first study describing the risk factors associated with INS A. nosocomialis bacteremia. Regulated antibiotic control policy, especially for carbapenem, and infection control measures targeting patients hospitalized in ICU, with chronic lung diseases and multiple invasive procedures, may be helpful in reducing INS A. nosocomialis infection.

Summary Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts.

Purpose: Bloodstream infections (BSIs) caused by Acinetobacter species have been extensively reported, however, which majorly focused on respiratory tract infections. The risk of mortality and the effect of early catheter removal on survival in catheter-related BSIs (CRBSIs) caused by Acinetobacter spp. remain unclear. This study aims to investigate that. Methods: This is a retrospective multicentric study conducted in Taiwan from 2012 to 2014. Patients with at least 1 positive blood culture and catheter culture for the same Acinetobacter spp., showing symptoms and signs of CRBSIs, were included (n = 119). Risk factors for 30-day mortality were analyzed using a logistic regression model. The characteristics of patients with early catheter removal (within 48 hours after CRBSIs) were compared to those without removal matching for age, sex, and disease severity. Results: There were no differences in 30-day mortality with regard to causative Acinetobacter spp., catheter type, site, and appropriateness of antimicrobial therapy. Patients with higher Acute Physiologic and Chronic Health Evaluation (APACHE) II scores (odds ratio [OR]: 1.12; 95% confidence interval [CI]: 1.02-1.23; P = .014), shock (OR: 6.43; 95% CI: 1.28-32.33; P = .024), and longer hospitalization before CRBSIs (OR: 1.04; 95% CI: 1.00-1.08; P = .027) had a significantly higher 30-day mortality rate. Early removal of catheters after CRBSIs was not associated with better survival benefits. Conclusion: Higher disease severity (APACHE II score), shock, and longer hospitalization before bacteremia were independently associated with a higher 30-day mortality in CRBSIs caused by Acinetobacter spp. In previous published guidelines, infected catheters were suggested to be removed in CRBSIs caused by gram-negative bacilli. Even though early removal of catheters did not associate with a better survival outcome in current results, it should be judiciously evaluated according to the clinical conditions and risks individually. For better elucidation of these issues, further well-controlled prospective study may be warranted.

The carbapenem breakpoints set by different organizations for Acinetobacter are discordant, but supporting clinical data are lacking. This study aimed to provide the first clinical outcome data to support the carbapenem breakpoints for Acinetobacter baumannii (Ab) group in patients with bacteremia. This study included 117 adults who received carbapenems for treatment of Ab group bacteremia in Taipei Veterans General Hospital over an 8-year period. We analyzed 30-day mortality rates among patient groups acquiring isolates with different carbapenem minimal inhibitory concentrations (MICs). The carbapenem MIC breakpoint derived from classification and regression tree (CART) analysis to delineate the risk of 30-day mortality was between MICs of ≤ 4 mg/L and ≥ 8 mg/L. Mortality rate was higher in patients acquiring isolates with carbapenem MIC ≥ 8 mg/L than ≤ 4 mg/L, by bivariate (54.9% [28/51] vs 25.8% [17/66]; P = 0.003) and survival analysis (P = 0.001 by log-rank test). Multivariate analysis using logistic regression and Cox regression models including severity of illness indices demonstrated that treating patients with Ab group bacteremia caused by isolates with a carbapenem MIC ≥ 8 mg/L with carbapenem was an independent predictor of 30-day mortality (odds ratio, 5.125; 95% confidence interval [CI], 1.946–13.498; P = 0.001, and hazard ratio, 2.630; 95% CI, 1.431–4.834; P = 0.002, respectively). The clinical outcome data confirmed that isolates with MIC ≤ 4 mg/L were susceptible to carbapenem, and those with MIC ≥ 8 mg/L were resistant in patients with Ab group bacteremia.

OBJECTIVES According to our previous study, OXA-58 translocates to the periplasm via the Sec pathway in carbapenem-resistant Acinetobacter baumannii (CRAb). In the present study, carbapenem-hydrolysing class D β-lactamases (CHDLs) belonging to the OXA-23, OXA-40 and OXA-51 families were examined to determine whether they are also Sec-dependent. Additionally, the effects of SecA inhibitors combined with carbapenems against CHDL-producing CRAb were examined. METHODS Cell fractionation and western blot analyses were performed to detect periplasmic His-tagged CHDLs. A chequerboard analysis with pairwise combinations of carbapenems (imipenem or meropenem) and SecA inhibitors (rose bengal, sodium azide or erythrosin B) was performed using six clinical CRAb isolates harbouring different CHDL genes. The fractional inhibitory concentration (FIC) index was determined. The combination with the lowest FIC index was subjected to a time-kill analysis to examine synergistic effects. RESULTS In an in silico analysis, the CHDLs OXA-23, OXA-40 and OXA-51 were preferentially translocated via the Sec system. The SecA inhibitor rose bengal decreased periplasmic translocation of His-tagged OXA-23 and OXA-83 (belonging to the OXA-51 family), but not OXA-72 (belonging to the OXA-40 family) from ATCC 15151 transformants. Imipenem or meropenem with rose bengal showed synergistic effects (FIC index, ≤0.5) for six and four clinical isolates, respectively. Imipenem or meropenem with sodium azide showed no interactions (FIC index, 0.5-4) against all clinical isolates. Imipenem and rose bengal had the lowest FIC index and showed synergy at 24 h in the time-kill assay. CONCLUSIONS Combinations of SecA inhibitors and carbapenems have synergistic effects against CHDL-producing CRAb.

Repeated isolation of multidrug-resistant Acinetobacter baumannii (MDRAB) from respiratory secretions poses a great challenge for infection control. We conducted a retrospective case-control study to evaluate the efficacy and adverse effect of inhaled colistin methanesulfonate (CMS) in the eradication of MDRAB from the respiratory tract. Patients who were admitted to Taipei Veterans General Hospital between February 2009 and June 2010, had at least two sets of monomicrobial culture of MDRAB from respiratory secretions, and remained in hospital for at least 14 days after the first isolation of MDRAB (index day) were included. Patients who received intravenous CMS were excluded. Patients who received CMS inhalation for ≥3 days were selected as cases whereas the controls were matched for age and Acute Physiology and Chronic Health Evaluation II score. Thirty-nine cases and controls were identified. The duration of CMS inhalation was 10.9 ± 3.6 days. The use of inhaled CMS was the only independent factor associated with the eradication of MDRAB within 14 days after the index day (OR 266.33; 95% CI 11.26–6302.18, p