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Funding: This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to G.F. and B.J.R., and by the University of St Andrews via a StARIS summer research bursary to S.K.L. The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV–VIS spectroscopy and LC–MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel. Publisher PDF

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of Ldt

Nosocomial infections caused by Acinetobacter baumannii currently represent a serious challenge for clinicians because treatment options are limited and frequently associated with significant toxicity. Cefiderocol is a first-in-class siderophore cephalosporin that has a proven efficacy for the treatment of multidrug-resistant Gram-negative infections, including carbapenem-resistant A. baumannii. The aim of this review is to evaluate the current evidence for the role of cefiderocol in the management of A. baumannii infections.In this review, we briefly summarize the available data on the efficacy (from randomized controlled trials) and on effectiveness and cure rates (from observational studies), pertaining to the use of cefiderocol for treatment of serious A. baumannii infections.Cefiderocol represents a promising and safe antibiotic option for treating patients with carbapenem-resistant A. baumannii infections. Due to conflicting mortality data from available experience, well-designed future randomized controlled trials and real-life studies are needed.

Acinetobacter baumannii causes healthcare-associated infections worldwide. Capsular polysaccharide (CPS) is shown an important virulence factor of A. baumannii both in vitro and in vivo. Capsule locus 2 (KL2) for CPS is the most common KL type and is associated with carbapenem resistance. It is unclear whether KL2 is related to the clinical outcome of invasive A. baumannii infection. Here we had followed patients with A. baumannii bacteraemia prospectively between 2009 and 2014. One-third of the unduplicated blood isolates were randomly selected each year for microbiological and clinical studies. The KL2 gene cluster was identified using polymerase chain reaction. A total of 148 patients were enrolled randomly. Eighteen isolates (12.2%) carried KL2, and 130 isolates (87.8%) didn't. Compared with non-KL2 isolates, KL2 isolates had significantly higher resistance to imipenem, sulbactam, and tigecycline. Compared with the non-KL group, in the KL2 group, the hospital stay before development of bacteraemia was longer (P

OBJECTIVE: To describe the course of the outbreak and infection control measures to stop the spread of sequence type 15 OXA-23-producing Acinetobacter baumannii in the Burn Center of Uppsala University Hospital, between November 2014 and the end of April 2015. METHODS: Compliance with hand hygiene, dress code, and cleaning routines were reviewed, the ward's environment was systematically investigated to identify potential environmental sources. Sampling routines for A. baumannii, from patients and environment, were established, and the epidemiological relationship was analysed for all carbapenem-resistant A. baumannii isolates using arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 54 patients were treated at the burn intensive care unit during the studied, approximately five months period, and an OXA-23-producing A. baumannii was isolated from nine patients (9/54, 17%), whereof two died (2/9, 22.2%). All isolates shared identical PFGE-genotype patterns and belonged to sequence type 15; AP-PCR was eligible for prompt epidemiological investigations. CONCLUSIONS: Higher awareness and increased compliance with hand hygiene and dress code as well as intensified cleaning protocols of the environment and equipment were successfully established and likely to have led to stop the spread of sequence type 15 OXA-23-producing Acinetobacter baumannii.

Intensive care unit (ICU) patients frequently have consequences from healthcare-associated infections (HAIs), which include bacteremia, pneumonia, urinary tract, skin, or soft tissue infections. . In this study, a total of 100 clinical specimens (urine, sputum and pus) were collected from patients admitted in the ICU. Results showed eighty two were positive growth culture ‎and admitted to the intensive care unit distributed 1 (1.2%)Klebsiella oxytoca,5(6.25%)‎Proteus mirabilis,2 (2.4%)‎Acinetobacter baumannii, 1 (1.2%)‎Serratia marcescensand 1 (1.2%)‎Burkholderia cepacia groupbacteria . The results of this study showed that the diagnostic test for16S rRNA‎bacteria (Proteus mirabilis), which numbered 5 (100%), bacteria ‎‎(Acinetobacterbaumannii), which numbered 2 (100%), bacteria ‎‎(Burkholderiacepacia), which numbered 1 (100%), bacteria ‎‎(Serratiamarcescens), which numbered 1 (100%), and Bacteria ‎‎(klebsiellaoxytoca), which numbered 1 (100%) after completing the ‎phenotypic and biochemical diagnosis diagnostic test (16S rRNA).

The main objective of this study was to identify carbapenem resistance mechanisms among clinical, commensal and environmental carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in 5 Tunisian intensive care units (ICUs).CRAB isolates were recovered from different sources: clinical specimens, rectal and environmental swabs. Bacterial identification was carried out using conventional methods and susceptibility testing according to EUCAST recommendations. Evaluation of phenotypic carbapenemase production of was performed using seven different methods, and molecular detection of carbapenemase-coding genes (blaAll in all, 46 CRAB isolates were identified in clinical specimens (n = 26/26), rectal swabs (n = 17/36) and environmental swabs (n = 3/63). Most of them (n = 41/46) were clonally related and found in the different ICUs. All of the CARB isolates harbored blaThe emergence and diffusion of clonal CRAB strains inducing high mortality rates in ICUs is a major public health concern. Enhanced infection control practices and mandatory staff education are needed to control the spread of these multidrug-resistant bacteria.

BACKGROUND: Viet Nam has high rates of antimicrobial resistance (AMR) but little capacity for genomic surveillance. This study used whole genome sequencing to examine the prevalence and transmission of three key AMR pathogens in two intensive care units (ICUs) in Hanoi, Viet Nam. METHODS: A prospective surveillance study of all adults admitted to ICUs at the National Hospital for Tropical Diseases and Bach Mai Hospital was done between June 19, 2017, and Jan 16, 2018. Clinical and environmental samples were cultured on selective media, characterised with MALDI TOF mass spectrometry, and sequenced with Illumina. Phylogenies based on the de-novo assemblies (SPAdes) were constructed with MAFFT (PARsnp), Gubbins, and RAxML. Resistance genes were detected with Abricate against the US National Center for Biotechnology Information database. FINDINGS: 3153 Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii isolates from 369 patients were analysed. Phylogenetic analysis revealed predominant lineages within A baumannii (global clone 2, sequence types ST2 and ST571) and K pneumoniae (ST15, ST16, ST656, ST11, and ST147) isolates. Isolation from stool was most common with E coli (87·0%) followed by K pneumoniae (62·5%). Of the E coli, 85·0% carried a blaCTX-M variant, while 81·8% of K pneumoniae isolates carried blaNDM (54·4%), or blaKPC (45·1%), or both. Transmission analysis with single nucleotide polymorphisms identified 167 clusters involving 251 (68%) of 369 patients, in some cases involving patients from both ICUs. There were no clear differences between the lineages or AMR genes recovered between the two ICUs. INTERPRETATION: This study represents the largest prospective surveillance study of key AMR pathogens in Vietnamese ICUs. Clusters of closely related isolates in patients across both ICUs suggests recent transmission before ICU admission in other health-care settings or in the community. FUNDING: UK Medical Research Council Newton Fund, Viet Nam Ministry of Science and Technology, Wellcome Trust, Academy of Medical Sciences, Health Foundation, and UK National Institute for Health and Care Research Cambridge Biomedical Research Centre.

In clinical practice, polymyxins are suggested to be used in combination with other antibiotics for improving their antibacterial efficacy and preventing the emergency of antibiotic-resistant strains. However, even though synergistic combination of polymyxin B with many antibiotics have been confirmed in various studies with different bacterial species and analyzing methods, which antibiotic is the best option for combination therapy of polymyxin B against MDR A. baumannii remains uncertain. In this study, we systematically analyzed the synergistic combination of polymyxin B with 12 other antibiotics against MDR A. baumannii isolated from a Chinese tertiary hospital using the checkerboard assay. The results suggest that, for polymyxin B-based combination therapy against MDR A. baumannii as characterized in this hospital, cefperazone-sulbactam may be the best partner, since it has the highest synergistic rate and the best synergistic effect with polymyxin B. Minocycline, imipenem, meropenem, ceftazidime, cefepime, amikacin and sulfamethoxazole also have some synergistic effects with polymyxin B, but piperacillin-tazobactam, ciprofloxacin, levofloxacin and tobramycin show no synergism. None of these 12 antibiotics has an antagonistic effect when combined with polymyxin B.

Recent emergence of high-level tigecycline resistance is mediated by tet(X) genes in Gram-negative bacteria, which undoubtedly constitutes a serious threat for public health worldwide. This study aims to identify tigecycline non-susceptible isolates and detect the presence of genes that are responsible for tigecycline resistance among local isolates in Iraq for the first time.Thirteen clinical isolates of Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa tigecycline non-susceptible were investigated from blood, sputum and burns specimens. The susceptibility of different antibiotics was tested by the VITEK-2 system. To detect tigecycline resistance genes, PCR was employed.Strains studied in this work were extremely drug-resistant and they were resistant to most antibiotic classes that were studied. The plasmid-encoded tet(X), tet(X1), tet(X2), tet(X3), tet(X4), tet(X5), tet(M) and tet(O) genes were not detected in the 13 isolates. The results showed that there is a clear presence of tet(A) and tet(B) genes in tigecycline non-susceptible isolates. All 13 (100%) tigecycline non-susceptible K. pneumoniae, A. baumannii and P. aeruginosa isolates harbored the tet(B) gene. In contrast, 4 (30.77%) tigecycline non-susceptible P. aeruginosa isolates harbored the tet(A) gene and there was no tigecycline non-susceptible A. baumannii isolate harboring the tet(A) gene (0%), but one (7.69%) tigecycline non-susceptible K. pneumoniae isolate harbored the tet(A) gene. A phylogenetic tree, which is based on the nucleotide sequences of the tet(A) gene, showed that the sequence of the local isolate was 87% similar to the nucleotide sequences for all the isolates used for comparison from GenBank and the local isolate displayed genetic diversity.According to this study, tet(B) and tet(A) play an important role in the appearance of tigecycline non-susceptible Gram-negative isolates.

The goal of this work was to systematically characterize and detect class 1, 2, and 3 integrons with many antibiotic resistance A. baumannii strains collected from a clinical environment in Iraq's Al-Muthanna hospitals. In this investigation, 24 non-replicated clinical strains of A. baumannii were evaluated using Chrome agar as a selective medium and PCR of the rplB gene. The clonal relatedness of the isolates to class 1 integron was evaluated using a PCR technique. The prevalence of class 1 integron was detected by PCR in only 12 clones of A. baumannii followed by HinfI digestion analysis showing three identical bands at 160 bp, 1350 bp, and 870 bp. In addition, PCR sequencing confirmed the presence of gene cassette arrays consisting of aacA4-catB8-aadA1 (100%) in class 1 integron. The sequence analysis of the integron shows 97.87 identity with A. baumannii isolates from Australia (GenBank accession number CP054302) among A. baumannii isolates. The blast analysis of this class I integron showed that the presence of the intI1, aacA4-catB8-aadA1 genes can considerably boost the acquisition of MDR phenotypes in A. baumannii isolates. We concluded that antibiotics of many types are widely used. The presence of integrons in A. baumannii is concerning for public health. In the clinical setting, it appears that the class 1 integron can be used as a predictive biomarker for the presence of MDR phenotypes. In these bacteria, however, the integron does not possess carbapenemases genes.

The K98 capsular polysaccharide (CPS) from the Acinetobacter baumannii clinical isolate, REV-1184, was studied by sugar analysis and Smith degradation along with one- and two-dimensional

Background: The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. Objective: The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). Methods: The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. Results: NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. Conclusion: The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

Two acylated forms of the higher sugar, 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid called pseudaminic acid, Pse5Ac7Ac and Pse5Ac7RHb where R indicates (R)-3-hydroxybutanoyl, have been found to occur in many capsular polysaccharide (CPS) types produced by isolates of an important human pathogen, Acinetobacter baumannii. The presence of either a psaABCEDF or psaABCGHF gene module at the K locus (KL) for CPS biosynthesis determines the type of the variant produced. Here, an A. baumannii clinical isolate 52-249, recovered in 2015 in Moscow, Russia, was found to include a novel psaABCIJF gene module in the KL218 sequence at the K locus. The CPS from 52-249 was extracted and studied by sugar analysis and partial acid hydrolysis along with one- and two-dimensional

Acinetobacter baumannii is an opportunistic pathogen known for high morbidity and mortality. It causes life-threatening infections, such as ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound and urinary tract infections (UTI). Increase in carbapenem resistance exhibited by A. baumannii has accentuated the need for novel targets for effective treatment. Despite the pronounced relevance of PPK2 as a pathogenicity determinant in several pathogens, it has not been explored as a drug target in A. baumannii. The present study was piloted to investigate the substrate binding by A. baumannii PPK2 (AbPPK2), a two-domain Class II polyphosphate kinase 2. A homology model of AbPPK2 was developed and validated for molecular docking of ATP and ADP in the predicted binding pocket. Further analysis of AbPPK2 revealed a set of common residues in the catalytic cleft interacting with ATP and ADP which would be useful for the screening of inhibitors against A. baumannii.

Introduction: Mortality of healthcare-associated Acinetobacter baumannii bacteremia can be 50-60% in intensive care units (ICUs). We aimed to determine the risk factors for 28-day mortality in patients with sepsis due to A. baumannii bacteremia during their ICU follow-up. Methodology: Demographic characteristics, disease severity scores on admission and bacteremia day (BD), resistance status, invasive interventions, and laboratory values showing the infection and severity of the BD, were compared between groups with and without mortality as a retrospective cohort study in the ICU of a tertiary hospital. Results: Of a total of 2411 patients, there were 192 cases of bacteremia. After applying the exclusion criteria, 39 patients were recruited for the study, 25 of whom died (mortality rate 64.1%). Higher age, Simplified Acute Physiology Score II (SAPS II) on admission and high Sequential Organ Failure Assessment Score (SOFA), Red Blood Cell Distribution Width (RDW) (p < 0.001), and C-Reactive Protein (CRP) (p = 0.002) on the BD and invasive intervention in follow-up were associated with mortality. When CRP and RDW were both positive, sensitivity was 72%, specificity was 100%, negative predictive value was 33%, and positive predictive value was 100% for the 28-day mortality after BD. Based on multivariate analysis, CRP and RDW values on the BD were independent risk factors for mortality. Conclusions: It is critical to monitor SOFA, RDW, and CRP values in older ICU patients with SAPS II scores and who undergo invasive intervention in follow-up. Increases in these parameters may indicate bacteremia with high mortality due to A. baumannii.

The incidence of liquefied petroleum gas (LPG)-related accidents in China has increased over the recent years. In addition, infection remains a big challenge in cases of severe burns. Therefore, the present study aimed to provide valuable information for a better control of infections in the event of such disasters. In this study, a total of 16 patients who suffered extremely severe burns after an LPG tanker explosion were included. Thereafter, bacteriological culture results were collected within a week. Of 16 patients, 13 (81.25%) were male and the average age of all patients was 60.63 years. In addition, the mean burned area was 83.03% TBSA. Additionally, a total of 553 organism cultures were conducted out of which 287 isolates (51.90%) showed positive results. Notably, 38.52% were Gram-negative bacteria, 7.59% were Gram-positive bacteria and 5.79% were fungi. Moreover, the most prevalent Gram-negative bacteria were Stenotrophomonas maltophilia (28.97%) followed by Acinetobacter baumannii (28.53%), and Klebsiella pneumoniae (14.02%). On the other hand, the three most predominant Gram-positive bacteria were Enterococcus faecalis (33.33%), Staphylococcus aureus (28.89%) and Staphylococcus sciuri (17.78%). Furthermore, the most common fungi included Candida (38.24%), Fusarium (20.59%) and Aspergillus fumigatus (14.71%). With regard to the bacterial resistance patterns, carbapenem-resistant organisms included Acinetobacter baumannii (97.80%), Pseudomonas aeruginosa (67.57%), and Klebsiella pneumoniae (75.56%). In addition, Staphylococcus sciuri, Staphylococcus epidermidis, and Staphylococcus haemolyticus were identified to be methicillin-resistant. This study revealed that there was a high incidence of infection in victims of severe burns as a result of mass burn accidents, accompanied by early fungal infection.

Carbapenem-resistant Acinetobacter baumannii (CRAB) sepsis is becoming an extreme threat caused by high-case fatality rates and poor prevention and control in ICUs worldwide. However, the risk of mortality among neonatal CRAB sepsis is still unclear.A retrospective medical records review study, which aimed to identify the risk factors of mortality in neonates with CRAB sepsis (including both bacteremia and/or meningitis) in Thailand from 1996 to 2019. All cases featuring positive blood and cerebrospinal fluid cultures for CRAB were reviewed. A multivariable logistic regression model was analyzed for nonsurvivors and survivors of neonatal CRAB sepsis.In a 24-year period, 47 of these were identified with CRAB sepsis. The median (interquartile range) gestational age and birth weight were 30 (28-35) weeks and 1500 (933-2482) g, respectively. The 30-day case fatality rate was 55% (26/47). In multivariable analysis, nonsurvivors of neonatal CRAB sepsis were associated with congenital heart disease (adjusted odds ratio [OR] = 1.33; 95% CI 1.06-1.66, P = 0.02), CRIB II score ≥9 (adjusted OR = 1.65; 95% CI: 1.20-2.27, P = 0.004), severe thrombocytopenia (adjusted OR = 1.45; 95% CI: 1.09-1.94, P = 0.02), and septic shock (adjusted OR = 1.62; 95% CI: 1.33-1.99, P0.001).The risk factors of mortality in neonates with CRAB sepsis are associated with congenital heart disease, CRIB II score ≥9, shock, and severe thrombocytopenia.

Bacteria in healthy body sites of humans and livestock waste may harbour antibiotic resistance and cause community-based opportunistic and resistant infections. The study profiled the antibiotic susceptibilities of resident bacteria in healthy humans and livestock waste. Gram-negative bacteria were isolated from 23 specimens including skin swabs (6), nasal swabs (4), urine (6), stool (3), chicken droppings (2) and cattle droppings (2). VITEK® 2 Automated System was used for identification, antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL) production test of the isolates. Nineteen (19) Gram-negative bacteria belonging to five genera and six species were identified, including Escherichia coli (n=9) 47.4%, Klebsiella pneumoniae ssp pneumoniae (n=1) 11.1%, Enterobacter cloacae ssp dissolvens (n=1) 11.1%, Acinetobacter baumannii (n=3) 15.8%, Acinetobacter haemolyticus (n=1) 11.1%, and Providentia stuartii (n=4) 21.1%. The isolates showed highest resistances to Ampicillin (78.6%) and Piperacillin (63.2%) and high susceptibilities to Ertapenem, Amikacin, Ciprofloxacin and Levofloxacin (100%); Ceftazidime, Cefepime, Meropenem (94.7%); Cefoxitin (93.3%); Gentamicin and Tobramycin (73.7%). Multiple Antibiotic Resistance (MAR) index values above the critical limit of 0.2 were shown by 100% (4/4) of Providentia stuartii isolates, 75% (3/4) of Acinetobacter isolates and 33.3% (3/9) of E. coli isolates. All the isolates tested negative for ESBL production. The public health implication is that resident bacteria from healthy individuals harbouring antibiotic resistance may transmit these to other bacteria or cause resistant opportunistic infections difficult to treat. Resistant bacteria from livestock can be transmitted to humans through the food chain. Proper disposal or decontamination of human body secretions and livestock waste is necessary.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a prevalent pathogen contributing to hospital infections. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and core-genome MLST (cgMLST) are frequently used methods to illuminate the nosocomial transmission of CRAB. In this study, we compared the discriminatory power of the three typing methods.Antimicrobial susceptibility tests were performed by the broth microdilution and Vitek2 methods. PFGE, MLST and cgMLST were conducted to determine the clonality and phylogenetic relationship of the strains. Whole-genome sequence data were acquired by an Illumina HiSeq 2000, and cgMLST was analysed by the Ridom SeqSphere+ v.7.2.3 software.A total of 149 carbapenem-resistant A. baumannii isolates had 15 different PFGE profiles (A-O type), and 73 of the isolates had related subtypes (A1 and A2), accounting for the majority of type A isolates. The maximum-likelihood phylogenetic analysis based on the cgMLST genes grouped the same PFGE clonal pattern A into nine different clusters. ST_Pasteur grouped all the strains into ST2, whereas ST_Oxford grouped the PFGE clonal pattern A isolates into six STs. In addition, the gdhB allele in the ST_Oxford scheme had two copies in five strains, which complicated the ST_Oxford typing.cgMLST was more discriminant than PFGE and MLST. CgMLST is the most suitable and comprehensive method for genotyping A. baumannii in surveillance and epidemiological research.

The superbug Acinetobacter baumannii is an increasingly prevalent pathogen of the intensive care units where its treatment is challenging. The identification of newer drug targets and the development of propitious therapeutics against this pathogen is of utmost importance. A drug target, cell division enzyme (FtsZ), involved in A. baumannii cytokinesis is a promising avenue for antibacterial therapy. Structure based virtual screening illustrated a lead-like molecule from Asinex antibacterial library to have the best binding affinity for the FtsZ active pocket. Computational pharmacokinetics predicted the compound to have the safest pharmacokinetics profile, thus maximizing the chances of the molecule reaching the market with enhanced efficacy and lesser toxicity. Molecular dynamics simulations in an aqueous environment revealed the flexibility of protein loop regions, and upward extension followed by the backward movement of the inhibitor N, N-dimethylpyridazin-3-amine ring on its axis. The active pocket residue Thr310 demonstrated to play significant role in inhibitor binding. The binding free energy predicted by MM/GBSA and MM/PBSA reflected system stability with a total value of -62.15 kcal/mol and -10.60 kcal/mol, respectively. The absolute binding free energy estimated by WaterSwap was -16 kcal/mol that validates and affirms complex stability. The inhibitor represents a promising scaffold as a lead optimization for the FtsZ enzyme.

Aims The aims were to determine the effects of subinhibitory concentrations of eight cephem and carbapenem antibiotics on the biofilm formation of Acinetobacter baumannii cells and examine their effects on pre-established biofilms. Methods and Results Effects of antibiotics on biofilm formation were assayed using microtitre plates with polystyrene peg-lids. Cefmetazole, ceftriaxone, ceftazidime and cefpirome increased the biomass of pre-established biofilms on pegs in the range of their sub-minimum inhibitory concentrations (MICs), whereas none increased biofilm formation by planktonic cells. Carbapenems had a negative effect. The constituents of antibiotic-induced biofilms were analysed. Ceftriaxone or ceftazidime treatment markedly increased the matrix constituent amounts in the biofilms (carbohydrate, 2.7-fold; protein, 8.9–12.7-fold; lipid, 3.3–3.6-fold; DNA, 9.1–12.2-fold; outer membrane vesicles, 2.7–3.8-fold and viable cells, 6.8–10.1-fold). The antibiotic-enhanced biofilms had increased outer membrane protein A and were resistant to the anti-biofilm effect of azithromycin. Conclusions Some cephems increased the biomass of pre-established biofilms in the ranges of their sub-MICs. The antibiotic-enhanced biofilms possessed more virulent characteristics than normal biofilms. Significance and Impact of the Study Incomplete administration of certain cephems following biofilm-related Ac. baumannii infections could adversely cause exacerbated and chronic clinical results.

Multidrug-resistant Acinetobacter baumannii (MDR-Ab), particularly strains producing oxacillinase (OXA)-type carbapenemases, have rapidly emerged in health care settings as a frequent cause of serious infections with limited treatment options. This study evaluated the in vitro activity of sulbactam (SUL) combined with durlobactam (DUR) against a collection of carbapenemase-producing A. baumannii, and investigated the mechanisms of resistance.Susceptibility testing was performed on 100 isolates by either broth microdilution or by the Epsilometer test. Isolates were screened for the insertion sequence ISAba1 upstream of the intrinsic chromosomal blaADC by polymerase chain reaction (PCR). Whole genome sequencing was performed on 25 SUL-DUR resistant isolates, and analyses were performed using the Center for Genomic Epidemiology platform. Target gene sequences were compared to A. baumannii American Type Culture Collection (ATCC) 17978.SUL-DUR exhibited excellent activity against A. baumannii isolates with susceptibility levels as follows: amikacin, 18%; colistin, 91%; cefepime, 5%; imipenem, 0%; minocycline, 46%; SUL, 3%; sulbactam-cefoperazone, 8%; SUL-DUR, 71% (based on a breakpoint at 4 mg/L). Twenty-five non-New Delhi metallo-ß-lactamase (NDM)-producing isolates had SUL-DUR MIC values4 mg/L, amongst which 14 isolates showed substitutions in penicillin-binding protein (PBP)3, previously shown to be associated with SUL-DUR resistance. Substitutions that have not previously been described were detected in SUL-DUR targets, namely PBP1a, PBP1b, PBP2, and PBP3. By contrast, there was no evidence of the involvement of permeability or efflux.SUL-DUR exhibited excellent in vitro antibacterial activity against carbapenemase-producing A. baumannii isolates. Amongst the 25 resistant isolates, we identified a number of mechanisms which may be contributing factors, in particular PBP substitutions and the production of specific beta-lactamases.

Acinetobacter baumannii is one of the most notorious nosocomial pathogens with high mortality rates. Recently, egg yolk antibody (IgY), has been considered as a promising biomolecule against pneumonia caused by this bacterium. Loop 3 of outer membrane protein 34 (Omp34) was predicted as a highly exposed immunogenic peptide. However, its immunogenicity remains to be experimentally elucidated. In the current study, a construct composed of 5 copies of loop3 of Omp34 labeled as Omp34L3X5 was designed. This construct as well as the recombinant Omp34 were expressed, purified, and injected into laying hens to raise specific antibodies. The specific IgYs were extracted from hyperimmune egg yolks. The Omp34L3X5 and whole cells (WC) of A. baumannii served as antigens in indirect ELISA to assess the purified IgYs reactivity. These antibodies were administered to neutropenic mice 1 h before the challenge with 10 × LD

Guidelines for treatment of resistant Acinetobacter baumannii (AB) are limited, leaving a knowledge gap in best practices for treatment. This study described treatments and outcomes of extensively-drug resistant (XDR) AB.Retrospective cohort study including patients with XDRAB (non-susceptible to at least 1 agent in all but 2 or fewer classes) and antibiotic treatment between 2012 and 2018 at Veterans Affairs Medical Centers. Descriptive statistics summarized antibiotics; propensity score adjusted regression models were fit to compare outcomes.Two hundred and seventy-six patients with 439 XDRAB cultures and Gram-negative targeted antibiotic treatment were included. One hundred and eighteen (43%) patients received monotherapy while 158 (57%) received combination therapy, most commonly including a carbapenem (n = 106, 67%) and polymyxin (n = 66, 42%). One hundred and eighty-four (67%) patients received inadequate treatment. In adjusted models, combination therapy did not decrease the odds of in-hospital (aOR 1.24, 95%CI 0.60-2.59) or 30-day (aOR 1.43, 95%CI 0.86-2.38) mortality, or median postculture length of stay (aIRR 1.11, 95%CI 0.86-1.43). Likewise, receipt of inadequate treatment was not associated with poorer outcomes.In this national cohort of patients with XDRAB, neither combination therapy nor receipt of adequate treatment improved outcomes. Further research is needed on optimal management of this difficult-to-treat pathogen with few effective antibiotic options.

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism’s adaptive efflux-mediated resistance phenotype.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Antibiotic-resistant Acinetobacter baumannii infections among patients in hospital settings are rising at an alarming rate. The World Health Organization has designated carbapenem-resistant A. baumannii as a priority pathogen for drug discovery. Based on the open drug discovery approach, we screened 400 compounds provided as a Pandemic Response Box by MMV and DNDi to identify compounds with antibacterial and antibiofilm activity against two A. baumannii reference strains using a highly robust resazurin assay. In vitro screening identified thirty compounds with MIC ≤ 50μM having growth inhibitory properties against the planktonic state. Five compounds, with MMV IDs MMV396785, MMV1578568, MMV1578574, MMV1578564, and MMV1579850, were able to reduce metabolically active cells in the biofilm state. Of these five compounds, MMV396785 showed potential antibacterial and antibiofilm activity with MIC, MBIC, and MBEC of 3.125 μM, 12.5, and 25–100 µM against tested A. baumannii strains, respectively, showing biofilm formation inhibition by 93% and eradication of pre-formed biofilms by 60–77.4%. In addition, MMV396785 showed a drastic reduction in the surface area and thickness of biofilms. Further investigations at the molecular level by qRT-PCR revealed the downregulation of biofilm-associated genes when exposed to 50 µM MMV396785 in all tested strains. This study identified the novel compound MMV396785 as showing potential in vitro antibacterial and antibiofilm efficacy against A. baumannii.

Acinetobacter baumannii (A. baumannii) is a highly problematic pathogen with an enormous capacity to acquire or upregulate antibiotic drug resistance determinants. The genomic epidemiology and resistome structure of 46 A. baumannii clinical isolates were studied using whole-genome sequencing. The isolates were chosen based on reduced susceptibility to at least three classes of antimicrobial compounds and were initially identified using MALDI-TOF/MS, followed by polymerase chain reaction amplification of blaOXA-51-like genes. The susceptibility profiles were determined using a broth microdilution assay. Multi-, extensive-, and pan-drug resistance was shown by 34.8%, 63.0%, and 2.2% of the isolates, respectively. These were most susceptible to colistin (95.7%), amikacin, and trimethoprim/sulfamethoxazole (32.6% each), while only 26.1% of isolates were susceptible to tigecycline. In silico multi-locus sequence typing revealed 8 Pasteur and 22 Oxford sequence types (STs) including four novel STs (STOxf 2805, 2806, 2807, and 2808). The majority of the isolates belonged to Global Clone (GC) 2 (76.4%), GC5 (19.6%), GC4 (6.5%), GC9 (4.3%), and GC7 (2.2%) lineages. An extensive resistome potentially conferring resistance to the majority of the tested antimicrobials was identified in silico. Of all known carbapenem resistance genes, blaOXA-23 was carried by most of the isolates (69.6%), followed by ISAba1-amplified blaADC (56.5%), blaNDM-1 and blaGES-11 (21.7% each), and blaGES-35 (2.2%) genes. A significant correlation was found between carbapenem resistance and carO mutations, which were evident in 35 (76.0%) isolates. A lower proportion of carbapenem resistance was noted for strains possessing both blaOXA-23- and blaGES-11. Amikacin resistance was most probably mediated by armA, aac(6′)-Ib9, and aph(3′)-VI, most commonly coexisting in GC2 isolates. No mutations were found in pmrABC or lpxACD operons in the colistin-resistant isolates. Tigecycline resistance was associated with adeS (N268Y) and baeS (A436T) mutations. While the lineage-specific distribution of some genes (e.g., blaADC and blaOXA-51-like alleles) was evident, some resistance genes, such as blaOXA-23 and sul1, were found in all GCs. The data generated here highlight the contribution of five GCs in A. baumannii infections in Egypt and enable the comprehensive analysis of GC-specific resistomes, thus revealing the dissemination of the carbapenem resistance gene blaOXA-23 in isolates encompassing all GCs.

Acinetobacter baumannii is the leading bacteria causative of nosocomial infections, with high fatality rates, mostly due to their multi-resistance to antibiotics. The capsular polysaccharide (k-type) is a major virulence factor. Bacteriophages are viruses that specifically infect bacteria and have been used to control drug-resistant bacterial pathogens. In particular, A. baumannii phages can recognize specific capsules, from a diversity of >125 that exist. This high specificity demands the in vivo identification of the most virulent A. baumannii k-types that need to be targeted by phage therapy. Currently, the zebrafish embryo has particularly attained interest for in vivo infection modeling. In this study, an A. baumannii infection was successfully established, through the bath immersion of tail-injured zebrafish embryos, to study the virulence of eight capsule types (K1, K2, K9, K32, K38, K44, K45, and K67). The model revealed itself as capable of discerning the most virulent (K2, K9, K32, and K45), middle (K1, K38, and K67), and the less virulent (K44) strains. Additionally, the infection of the most virulent strains was controlled in vivo resorting to the same technique, with previously identified phages (K2, K9, K32, and K45 phages). Phage treatments were able to increase the average survival from 35.2% to up to 74.1% (K32 strain). All the phages performed equally well. Collectively, the results show the potential of the model to not only evaluate virulence of bacteria such as A. baumannii but also assess novel treatments' effectiveness., The authors acknowledge the support from Ivone Pinheiro and Sofia Azevedo in the zebrafish culturing and breeding and from Alejandro Garrido-Maestu in the A. baumannii culture and quantification methodology. B.E. acknowledges the financial support of the project SbDtoolBox— Nanotechnology-based tools and tests for Safer-by-Design nanomaterials, with the reference n° NORTE-01-0145-FEDER-000047, funded by Norte2020—North-Regional Operational Programme under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

(1) Background: Acinetobacter baumannii has become the most important pathogen responsible for nosocomial infections in health systems. It expresses several resistance mechanisms, including the production of β-lactamases, changes in the cell membrane, and the expression of efflux pumps. (2) Methods: A. baumannii was detected by PCR amplification of the blaOXA-51-like gene. Antimicrobial susceptibility to fluoroquinolones and aminoglycosides was assessed using the broth microdilution technique according to 2018 CLSI guidelines. Efflux pump system activity was assessed by the addition of a phenylalanine–arginine beta-naphthylamide (PAβN) inhibitor. (3) Results: A total of nineteen A. baumannii clinical isolates were included in the study. In an overall analysis, in the presence of PAβN, amikacin susceptibility rates changed from 84.2% to 100%; regarding tobramycin, they changed from 68.4% to 84.2%; for nalidixic acid, they changed from 73.7% to 79.0%; as per ciprofloxacin, they changed from 68.4% to 73.7%; and, for levofloxacin, they stayed as 79.0% in both groups. (4) Conclusions: The addition of PAβN demonstrated a decrease in the rates of resistance to antimicrobials from the family of quinolones and aminoglycosides. Efflux pumps play an important role in the emergence of multidrug-resistant A. baumannii strains, and their inhibition may be useful as adjunctive therapy against this pathogen.

Introduction: We sought to evaluate secondary infections (SIs) in patients admitted to the intensive care unit (ICU) for COVID-19 with respect to incidence, causative pathogens, and clinical outcomes. Methodology: In this two-centre retrospective study, we analysed 146 patients (96 males, 50 females; median age, 64 years) admitted to the ICU with COVID-19 between March 26 and December 31, 2020. Inclusion criteria were an ICU admission for at least 48 hours and age beyond 18 years. Patients with and without SIs were compared and the impacts of SIs and carbapenem resistance on mortality were analysed. Results: During ICU admission, 84 episodes of SIs developed in 58 patients (39.7%). A total of 104 isolates were recovered, with Gram-negative bacteria most frequent accounting for 74%. At least one carbapenem-resistant pathogen (n = 61) was recovered in 41 patients (70.1%). In multivariate analysis, the use of ECMO and an elevated procalcitonin level were significantly associated with the development of SIs. The mortality rate and the incidence of carbapenem resistance did not differ significantly in COVID-19 patients with and without SIs (p = 0.059 and p = 0.083, respectively). Conclusions: The incidences of SIs and carbapenem resistance among COVID-19 patients were alarming, emphasizing stricter infection control measures in the ICU setting. © 2022 Journal of Infection in Developing Countries. All rights reserved.

Bacterial resistance is a problem that is giving serious cause for concern because bacterial strains such as Acinetobacter baumannii and Pseudomonas aeruginosa are difficult to treat and highly opportunistic. These bacteria easily acquire resistance genes even from other species, which confers greater persistence and tolerance towards conventional antibiotics. These bacteria have the highest death rate in hospitalized intensive care patients, so strong measures must be taken. In this review, we focus on the use of antimicrobial peptides (AMPs) as an alternative to traditional drugs, due to their rapid action and lower risk of generating resistance by microorganisms. We also present an overview of beta-lactams and explicitly explain the activity of AMPs against carbapenemase-producing bacteria as potential alternative agents for infection control.

With the increasingly serious drug resistance of Acinetobacter baumannii, there is an increasingly urgent need for new antibacterial drugs. Phage lysin PlyAB1 has a bactericidal effect on drug-resistant A. baumannii, which has the potential to replace antibiotics to fight infection caused by A. baumannii. However, its application is limited by its thermal stability and lytic activity. To solve these problems, molecular dynamics (MD) simulations combined with Hotspot wizard 3.0 were used to identify key residue sites affecting thermal stability, and evolutionary analysis combined with multiple sequence alignment was used to identify key residue sites affecting lytic activity. Four single-point variants with significantly increased thermal stability and four single-point variants with significantly lytic activity were obtained, respectively. Furthermore, by superimposing mutations, we obtained three double-point variants, G100Q/K69R, G100R/K69R, and G100K/K69R, with significantly improved thermal stability and improved lytic activity. At 45°C, the lytic activity and half-life of the optimal variant G100Q/K69R were 1.51- and 24-fold higher than those of the wild PlyAB1, respectively. These results deepen our understanding of the structure and function of phage lysin and contribute to the application of phage lysin in antibiotic substitution.

Aim: To control the spread of Acinetobacter baumannii in hospitals, it is necessary to identify the reservoir of organisms and the way they are transmitted. This study analyzed samples by BOX-PCR and enterobacterial repetitive intergenic consensus PCR techniques. Methods: Isolated strains were identified using the Microgen kit and blaOXA-51 gene. The genetic diversity of strains that were sensitive or resistant to colistin was evaluated by BOX-PCR and enterobacterial repetitive intergenic consensus PCR methods. Results: A total of 13% of the isolates were resistant to colistin, whereas 87% of the strains were sensitive to this medication. A. baumannii strains that were resistant or sensitive to colistin were divided into five groups using the BOX-PCR method and six groups using the enterobacterial repetitive intergenic consensus PCR method. Conclusion: Rapid identification and the use of appropriate tools to control colistin-resistant clones are essential to prevent the further spread of A. baumannii.

Introduction: Secondary Bacterial Infections (SBIs) of the respiratory system are one of the biggest medical concerns in patients undergoing hospitalization with a diagnosis of COVID-19. This study aims to provide relevant data for the initiation of appropriate empirical treatment after examining the etiology and antimicrobial resistance of SBIs in COVID-19 patients under care in the Intensive Care Units (ICUs) in the largest pandemic hospital of our country. Methodology: Between March 16, 2020 and December 31, 2021, 56,993 COVID patients were hospitalized, of which 7684 were admitted to ICUs. A total of 1513 patients diagnosed with SBIs have been included in this study. During the course of the study, demographic data, clinical course, etiology and antimicrobial resistance data of all patients were collected. Results: The most common causative agents of SBIs were inferred as Acinetobacter baumanii (35.1%), Staphylococcus aureus (15.2%), Klebsiella pneumoniae (12.3%) and Pseudomonas aeruginosa (10.4%). The isolation rates of carbapenem-resistant and colistin-resistant A. baumannii, K. pneumoniae and P. aeruginosa were 83.7%; 42.7%, 79.2%, and 5.6%, 42.7%, 1.7%, respectively. Acinetobacter pittii clustering was seen in one of the ICUs in the hospital. Multidrug resistant 92 (5.4%) Corynebacterium striatum isolates were also found as a causative agent with increasing frequency during the study period. Conclusions: SBI of the respiratory system is one of the major complications in patients hospitalized with COVID-19. The antimicrobial resistance rates of the isolated bacteria are generally high, which indicates that more accurate use of antibacterial agents is necessary for SBIs in patients hospitalized with COVID-19 diagnosis.

Background To date, no real-world data are available to describe cefiderocol use in carbapenem-resistant Acinetobacter baumannii (CRAB) meningitis. Furthermore, cefiderocol pharmacokinetic (PK) data to support CNS penetration in human subjects are limited. These gaps pose a significant concern for clinicians who are faced with treating such infections when considering cefiderocol use. Objectives To describe cefiderocol CSF and plasma PK and pharmacodynamic (PD) data from two different dosing regimens [2 g IV q6h (regimen 1) and 2 g IV q8h (regimen 2)] during treatment of CRAB meningitis. Patients and methods A 61-year-old woman with CRAB meningitis was treated with cefiderocol and intraventricular gentamicin. Steady-state plasma and CSF cefiderocol concentrations were evaluated on Day 19 (regimen 1) and Day 24 (regimen 2) during the cefiderocol treatment course. Results CSF AUC was 146.49 and 118.28 mg·h/L, as determined by the linear-log trapezoidal method for regimens 1 and 2, respectively. Penetration into CSF estimated as the AUCCSF/AUCfree plasma ratio was 68% and 60% for regimens 1 and 2, respectively. Estimated free plasma and CSF concentrations exceeded the MIC of the isolate for 100% of the dosing interval. Microbiological and clinical cure were achieved, and no cefiderocol-associated adverse effects were observed. Conclusions Cefiderocol, when given as 2 g q8h and 2 g q6h, attained CSF concentrations that exceeded the organism-specific MIC and the CLSI susceptible breakpoint (≤4 mg/L) for 100% of the dosing interval.

Introduction : Clinical use of mobile phones have increased exponentially. Whilst evidence of contamination is documented, a key factor when determining potential risks of contamination, is establishing the duration the organism remains viable on the device. If pathogens are found to persist for extended duration, healthcare mobile phones may become fomites for cross departmental transmission. Aim : Determine the duration pathogenic bacteria, Acinetobacter baumannii, Escherichia coli, two Pseudomonas sp. Bacillus cereus, Enterococcus faecalis susceptible and resistant to vancomycin (VSE and VRE) Staphylococcus aureus susceptible and resistant to methicillin (MSSA and MRSA), and a coagulase negative Staphylococcus (CoNs) can remain viable on a mobile phone under controlled conditions. Method : Phones were inoculated with 106 – 107 of each bacterium. The duration of viability was measured from the point the inoculum had dried and CFUs retrieved at timed intervals over 28 days. Results : The mean percentage of bacteria viable at each time point was significantly different (20mins, p=0.004, 1hour p=0.014, 6hours p = 0.006, 24hours p=0.004, 7 days p = 0.007, 14 days p = 0.003, 21 days p = 0.002- and 28-days p =0.004). Gram-positive bacteria remained viable longer than gram-negative bacteria (p=0.010). MSSA declined faster than MRSA within the first 6 hours (p=0.036). Conclusion : The extended duration of bacterial viability indicates the ability for pathogens to persist on a device and remain viable long enough to be transmitted to new areas both within the hospital and out to the community. Mobile phone decontamination should occur in combination of hand hygiene.

Multidrug-resistant Acinetobacter baumannii (A. baumannii) infections are becoming more prevalent all over the world. As a cost-effective and preventative method, vaccination seems to be required against this bacterium. In the present study, subtractive proteomics along with reverse vaccinology approaches was used to predict suitable therapeutics against A. baumannii. Using the Vaxign online tool, we studied over 35 genomes of A. baumannii strains and chose outer membrane and secreted proteins of A. baumannii 1656-2 as possible vaccine candidates. Then, investigations were performed on the immunogenicity, antigenic characteristics, physicochemical properties, B-cell and MHC class I, and MHC class II molecules epitope densities of proteins. After optimizing the codon of the proteins, the pcDNA3.1( +) expression construct was designed and the immunogenicity, allergenicity, and physicochemical properties of the vaccine construct were predicted. Hcp and OmpC proteins were predicted as extracellular and outer membrane proteins, respectively. These proteins interact with 10 other proteins to form a network of protein interactions with virulence properties. Immunoassays of Hcp and OmpC proteins showed antigenicity of 0.88 and 0.79, respectively. These proteins have 5 structural cell epitope points and 5 linear B epitope points. They are also able to bind to different HLA alleles of MCH class I/class II as selected immunogenic proteins and designed non-allergenic structures with solubility of 0.650 and immunogenicity score of 0.91. The results of this "in silico" study indicate high specificity and the development of a significant humoral and cellular immune response. It can be concluded that the Hcp and OmpC dual vaccine construct is one of the promising candidates against A. baumannii. The findings of this "in silico" study show excellent specificity and the emergence of a substantial humoral and cellular immune response. This is a computer-based study that needs to be tested in vitro and in vivo to corroborate the conclusions of the vaccine design procedures.

Healthcare associated meningitis and ventriculitis (HCAMV) are serious complications of neurosurgical procedures. We conducted a retrospective cohort study of patients with HCAMV treated at the University Hospital for Infectious Diseases Zagreb during the 2013–2019 period. A total of 144 patients with 151 episodes of HCAMV were included. The most common indications for neurosurgical procedures were brain tumor, hemorrhage and hydrocephalus. Etiology was identified in 90 (59.6%) episodes (either positive CSF culture or positive PCR), and in other 61 (40.39%) the diagnosis of HCAMV was made based on clinical and CSF parameters, without microbiologic confirmation. Carbapenem-resistant Acinetobacter baumannii was the most common pathogen (15.89%), followed by Staphylococcus aureus (13.91%), Pseudomonas aeruginosa (13.25%) and Coagulase negative staphylococci (7.95%). Overall, 24 (16.3%) patients died, and the majority had adverse outcomes, persistent vegetative state (8, 5.56%) and severe disability (31, 21.53%). The worst clinical outcomes were observed in A. baumannii infections. High rate of complications, the need for external ventricular drainage (re)placement often complicated with nosocomial infections and prolonged stay in intensive care units were observed. Clinicians should be aware of local microbial epidemiology on guiding proper empirical antimicrobial treatment in patients with HCAMV.

The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO

Antimicrobial drug resistance is one of the major threats to global health. It has made common infections increasingly difficult or impossible to treat, and leads to higher medical costs, prolonged hospital stays and increased mortality. Infection rates due to multidrug-resistant organisms (MDRO) are increasing globally. Active agents against MDRO are limited despite an increased in the availability of novel antibiotics in recent years. This guideline aims to assist clinicians in the management of infections due to MDRO. The 2019 Guidelines Recommendations for Evidence-based Antimicrobial agents use in Taiwan (GREAT) working group, comprising of infectious disease specialists from 14 medical centers in Taiwan, reviewed current evidences and drafted recommendations for the treatment of infections due to MDRO. A nationwide expert panel reviewed the recommendations during a consensus meeting in Aug 2020, and the guideline was endorsed by the Infectious Diseases Society of Taiwan (IDST). This guideline includes recommendations for selecting antimicrobial therapy for infections caused by carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Enterobacterales, and vancomycin-resistant Enterococcus. The guideline takes into consideration the local epidemiology, and includes antimicrobial agents that may not yet be available in Taiwan. It is intended to serve as a clinical guide and not to supersede the clinical judgment of physicians in the management of individual patients.

Objectives To explore the in vitro antimicrobial susceptibility among clinically important Gram-negative bacteria (GNB) in Taiwan. Methods From 2016 through 2018, a total of 5458 GNB isolates, including Escherichia coli (n = 1545), Klebsiella pneumoniae (n = 1255), Enterobacter species (n = 259), Pseudomonas aeruginosa (n = 1127), Acinetobacter baumannii complex (n = 368), and Stenotrophomonas maltophilia (n = 179), were collected. The susceptibility results were summarized by the breakpoints of minimum inhibitory concentration (MIC) of CLSI 2020, EUCAST 2020 (for colistin), or published articles (for ceftolozane/tazobactam). The resistance genes among multidrug-resistant (MDR) or extensively drug-resistant (XDR)-GNB were investigated by multiplex PCR. Results Significantly higher rates of non-susceptibility (NS) to ertapenem and carbapenemase production, predominantly KPC and OXA-48-like beta-lactamase, were observed in Enterobacterales isolates causing respiratory tract infection than those causing complicated urinary tract or intra-abdominal infection (12.7%/3.44% vs. 5.7%/0.76% or 7.7%/0.97%, respectively). Isolates of Enterobacter species showed higher rates of phenotypic extended-spectrum β-lactamase and NS to ertapenem than E. coli or K. pneumoniae isolates. Although moderate activity (54–83%) was observed against most potential AmpC-producing Enterobacterales isolates, ceftolozane/tazobactam exhibited poor in vitro (44.7–47.4%) activity against phenotypic AmpC Enterobacter cloacae isolates. Additionally, 251 (22.3%) P. aeruginosa isolates exhibited the carbapenem-NS phenotype, and their MDR and XDR rate was 63.3% and 33.5%, respectively. Fifteen (75%) of twenty Burkholderia cenocepacia complex isolates were inhibited by ceftolozane/tazobactam at MICs of ≤4 μg/mL. Conclusions With the increase in antibiotic resistance in Taiwan, it is imperative to periodically monitor the susceptibility profiles of clinically important GNB.

Humanity is facing an enormous and growing worldwide threat from the emergence of multi-drug-resistant Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Polymyxin B and E (colistin) constitute the last-line therapies for treating multi-drug-resistant Gram-negative bacteria. Polymyxin is a cationic antibacterial peptide that can destroy the outer membrane of Gram-negative bacteria. With the increasing clinical application of polymyxin, however, there have been many reports of the occurrence of polymyxin-resistant Gram-negative bacteria. This resistance is mainly mediated by the modification or complete loss of lipopolysaccharide (LPS). LPS is also a virulence factor of Gram-negative bacteria, and alterations of LPS may correlate with virulence. Although it is generally believed that the biological costs associated with drug resistance may enable benign susceptible bacteria to overcome resistant bacteria when antibiotic pressure is reduced, some studies have shown that polymyxin-resistant bacteria are associated with higher virulence and greater fitness compared with their susceptible counterparts. To predict the development of polymyxin resistance and evaluate interventions for its mitigation, it is important to understand the relative biological cost of polymyxin resistance compared with susceptibility. The impact of polymyxin resistance mechanisms on the virulence and fitness of these three Gram-negative bacteria are summarized in this review.

Background: The diminishing antimicrobial options for the treatment of XDR and PDR Acinetobacter baumannii is an increasing concern. In this study, we assessed the in vitro synergy of the fosfomycin (FOS) with meropenem (MEM), amikacin (AK), tigecycline (TGC), and colistin (CL) in whole genome sequenced isolates. Methods: Non-replicate whole genome sequenced (illumina next-generation sequencing platform, Clevergene, India), A. baumanii (7 XDR, 1PDR) were subjected to in vitro synergy testing by checkerboard (CB) and time kill assay (TKA) after MIC determination, with glucose-6-phosphate being incorporated in all runs. FOS was used as a cornerstone drug in four combinations and colistin in one. ResFinder, MLST, PlasmidFinder, and CSIPhylogeny tools were used. Results: Mortality occurred in three patients. Diverse MLST were observed, ST-1962 (3 isolates) and one each of ST2062, ST2063, ST1816, ST1806, ST234. FOS MICs ranged from 32 to 128 mg/L, MEM MIC: 16–64 mg/L, TGC MIC: ≤2–≤4 mg/L and AK MIC: >512 mg/L. CL: MIC range, 0.25–≤2 mg/L, PDR MIC > 16 mg/L. Synergy results by CB: FOS-MEM: synergy in ⅞ (90%) isolates. Synergy lowered MEM MICs to susceptibility breakpoints in 6/8 cases. CL-MEM: Excellent synergy (3/3) isolates. FOS-AK: Indifference in ⅞, antagonism ⅛ (AK-susceptible isolate). FOS-TGC: Partial synergy (PS) in 8/8 (TGC MIC dropped to ≤0.25 mg/L in 3/8). In the PDR isolate, synergy was seen in FOS-MEM, CL-MEM, PS in FOS-CL, FOS-TGC, indifference in FOS-AK. TKA: Excellent synergy was observed with FOS-MEM from 4 h, while FOS-AK and FOS-TGC demonstrated synergy at 24 h. Synergy was achieved despite presence of widespread resistance markers against aminoglycosides (AacAad, AadA, AadB, Aph3″Ia, ArmA, Arr, StrA, StrB), beta-lactams (ADC, BlaA1, BlaA2, Zn-dependent_hydrolase, OXA-23, OXA-51, PER-1,TEM-1D, CARB-5, Mbl), sulphonamides (SulII, SulI), phenicols (CatBx, CmlA), macrolides (MphE, MsrE) and tetracycline (TetB) were widespread. Carbapenemase, CARB-5 was present in one isolate. Beta-lactamase genes OXA-23, OXA-51, BlaA2, Zn-dependent_hydrolase, ADC, Mbl and macrolide resistance genes MphE, MsrE were present in all 8 isolates. Conclusions: FOS-MEM and CL-MEM are promising combinations against A. baumannii. Synergy of FOS-MEM in intrinsically resistant A. baumannii shows that this antibiotic combination might be useful in treating such XDR and PDR pathogens.

The mechanism of tigecycline resistance in A. baumannii remains largely unclear. In this study, we selected a tigecycline-resistant and a tigecycline-susceptible strain from a tigecycline-susceptible and a resistant strain, respectively. Proteomic and genomic analyses were performed to elucidate the variations associated with tigecycline resistance. Our study showed proteins associated with efflux pump, biofilm formation, iron acquisition, stress response, and metabolic ability are upregulated in tigecycline resistant strains, and efflux pump should be the key mechanism for tigecycline resistance. By genomic analysis, we found several changes in the genome that can explain the increased level of efflux pump, including the loss of the global negative regulator hns in the plasmid and the disruption of the hns gene and acrR gene on the chromosome by the insertion of IS5. Collectively, we not only revealed the phenomenon that the efflux pump is mainly responsible for tigecycline resistance, but also highlighted the mechanism at the genomic level, which will help in understanding the resistance mechanism in detail and provide clues for the treatment of clinical multiple drug-resistant A. baumannii.

Antimicrobial resistance poses a global threat to public health. Of great concern are Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales with resistance to carbapenems or third-generation cephalosporins. The aim of the present study was to investigate the in vitro activity of the novel siderophore cephaloporin cefiderocol (CID) and four comparator β-lactam-β-lactamase-inhibitor combinations and to give insights into the genetic background of CID-resistant isolates. In total, 301 clinical Enterobacterales and non-fermenting bacterial isolates were selected for this study, including randomly chosen isolates (set I, n = 195) and challenge isolates (set II, n = 106; enriched with ESBL and carbapenemase producers, as well as colistin-resistant isolates). Isolates displayed CID MIC50/90 values of 0.12/0.5 mg/L (set I) and 0.5/1 mg/L (set II). Overall, the CID activity was superior to the comparators against A. baumannii, Stenotrophomonas maltophilia and set II isolates of P. aeruginosa. There were eight CID-resistant isolates detected (MIC > 2 mg/L): A. baumannii (n = 1), E. cloacae complex (n = 5) and P. aeruginosa (n = 2). Sequencing analyses of these isolates detected the acquired β-lactamase (bla) genes blaNDM-1,blaSHV-12 and naturally occurring blaOXA-396, blaACT-type and blaCMH-3. In conclusion, CID revealed potent activity against clinically relevant organisms of multidrug-resistant Enterobacterales and non-fermenters.

The use of synergistic combinations between natural compounds and commercial antibiotics may be a good strategy to fight against microbial resistance, with fewer side effects on human, animal and environmental, health. The antimicrobial capacity of four compounds of plant origin (thymol and gallic, salicylic and gentisic acids) was analysed against 14 pathogenic bacteria. Thymol showed the best antimicrobial activity, with MICs ranging from 125 µg/mL (for Acinetobacter baumannii, Pasteurella aerogenes, and Salmonella typhimurium) to 250 µg/mL (for Bacillus subtilis, Klebsiella aerogenes, Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus, and Streptococcus agalactiae). Combinations of thymol with eight widely used antibiotics were studied to identify combinations with synergistic effects. Thymol showed synergistic activity with chloramphenicol against A. baumannii (critical priority by the WHO), with streptomycin and gentamicin against Staphylococcus aureus (high priority by the WHO), and with streptomycin against Streptococcus agalactiae, decreasing the MICs of these antibiotics by 75% to 87.5%. The kinetics of these synergies indicated that thymol alone at the synergy concentration had almost no effect on the maximum achievable population density and very little effect on the growth rate. However, in combination with antibiotics at the same concentration, it completely inhibited growth, confirming its role in facilitating the action of the antibiotic. The time–kill curves indicated that all the combinations with synergistic effects were mainly bactericidal.