Baumannii Group Research Articles

METHOD FOR PHENOTYPIC IDENTIFICATION OF ACINETOBACTER SEIFERTII BACTERIA

Abstract. The aim of the study is to increase the accuracy of Acinetobacter seifertii species identification by the phenotypic method. Study objects: 5 strains of A. seifertii (3 isolated in the microbiological laboratory at the Military Medical Academy, 2 strains obtained from the Pasteur Institute (St. Petersburg)). Clinical strains of the A. baumannii group (Ab group) isolated at the Military Medical Academy in 2023-2024 were also studied: A. baumannii (n=152), of which 37 strains of the tryptophandestruens biovar, A. nosocomialis (n=12), A. pittii (n=6), 3 strains of A. calcoaceticus from the Neva River. The type of such strains was determined by MALDI-TOF mass spectrometry. The belonging of the strains to the Ab group was established by taxonomic tests. The biovar A. baumannii bv. tryptophandestruens was determined by a chromogenic reaction on sodium benzoate-containing nutrient medium. Urease of rapid activity was detected by a micro-volumetric method assessed 3 hours later. Utilization of D-xylose as the only carbon source was determined on a nutrient medium containing (g/l): D-xylose 2.0; NH4CI 5.0; NH4NO3 1.0; Na2SO4 2.0; K2HPO4 3.0; KH2PO4 1.0; MgSO4 0.1; bromothymol blue 1.6% aqueous solution 4 ml; bacteriological agar 15.0; distilled water 1 l; pH 7.2 ± 0.2; sterilization at 112oC 20 min. A pure bacterial culture was studied, in which signs of the Ab group and growth on a sodium acetate-containing nutrient medium were previously detected (control of no auxotrophy). The agar bacteria culture was suspended in 0.1 ml of 0.85% sodium chloride solution, one loop of bacterial suspension was sown with a stroke on a nutrient medium with D-xylose and a medium without D-xylose (control), incubated aerobically at 30oC 24-48 hours. The absence of bacterial growth on a medium with D-xylose and a medium without D-xylose in the presence of their growth on a nutrient medium with sodium acetate indicates belonging to the species A. seifertii. Studies showed that all A. seifertii strains do not utilize D-xylose, and all strains of other species of the Ab group utilize D-xylose. A. seifertii bacteria do not utilize L-arabinose, however, 29.6±3,7% of A. baumannii strains do not utilize L-arabinose, including all 37 strains of the tryptophandestruens biovar, which determines the unreliability of this test for the identification of A. seifertii. Most strains of A. seifertii (4 out of 5) have a urease of rapid activity, which suggests their proximity to the species A. nosocomialis. A method has been developed for identification of A. seifertii bacteria based on a set of phenotypic features of Ab group bacteria with a test for D-xylose utilization. It has been established that the use of the D-xylose utilization test in conjunction with the detection of rapid urease activity allows to accurately identify the bacteria A. seifertii and A. nosocomialis among other species of the Ab group.

Characteristics of antibiotic sensitivity of Acinetobacter baumannii bv. tryptophandestruens and Acinetobacter baumannii clinical isolates

The aim of the study was to determine the sensitivity of the bacteria of the biovar Acinetobacter baumannii bv. tryptophandestruens to antibiotics used to treat Acinetobacter-infection. The object of the study was 86 primary clinical isolates of A. baumannii, of which 34 strains of A. baumannii bv. tryptophandestruens isolated in the microbiological laboratory of the Military Medical Academy in 2021–2022. Species identification of bacteria was carried out by MALDI-ToF mass spectrometry. The biovar tryptophandestruens of A. baumannii was determined on a dense chromogenic medium. The sensitivity of A. baumannii isolates to antibiotics was determined by a “Vitek 2 compact” microbiological analyzer (bioMerieux, France). Clinical categories of isolates sensitivity to antibiotics (S) were identified based on the boundary values of minimum suppressive concentrations (MSC, mg/L) established by the European Committee for the Determination of Sensitivity to Antimicrobial Preparates (EUCAST), version 11.0. Isolates of A. baumannii bv. tryptophandestruens and groups of isolates of other A. baumannii very rarely sensitive to meropenem (8.8±4.6%; 3.8±2.6%, respectively, p 0.05), ciprofloxacin (5.8±4.0; 9.6±4.1%, p 0.05). Strains sensitive to gentamicin are rarely represented in both groups (26.7±7.55; 19.3±5.5%, p 0.05) trimethoprim/sulfamethoxazole (38.8±8.3%; 23.6±5.3%, p 0.05). Sensitivity to polymyxin B was preserved in all strains of both groups (100%). Strains of the tryptophandestruens biovar A. baumannii group surpass strains of the other strain of A. baumannii group in the frequency of isolates with MSC50% tigecycline (MSC50% — 0.5 mg/l; MSC50% — 2 mg/l, respectively), and also have significantly more strains with MSC 0.5 mg/l (52.9% and 15.4%, respectively, p 0.01 ). Thus, the clinical isolates of the biovar A. baumannii bv. tryptophandestruens have no significant differences with isolates of other strains of A. baumannii in the frequency of strains sensitive (S) to meropenem, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, polymyxin B, but they differ in terms of sensitivity to tigecycline — a higher frequency of strains with an MSC of 0.5 mg/l (p 0.01) and an MSC of 50%.

Phylogenomics of Acinetobacter species and analysis of antimicrobial resistance genes.

Non-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies. Phylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment. The Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species. In conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.

Defining the Risk Factors for the Evolution of Pan-Drug Resistance (PDR) Acinetobacter baumanni Infections in Intensive Care Units

ABSTRACT
 Intorductıon:Acinetobacter baumannii is one important nosocomial pathogenes. Acinetobacter infections causes long in hospital stay, mortality and morbidity. The aim of this study is to define the risk factors of PDR A. baumannii caused health care related(HCR) infections. 
 
 Materyal and Methods:In the study of Cumhuriyet University Hospital between 01.01.201231.12.2013 is a case-control study was performed retrospectively. 49 PDR A. baumannii caused ventilator associated pneumonia and bacteraemia, 71 other bacteria caused ventilator associated pneumonia and bacteraemia patients were involved in this study. The PDR A. baumannii infection observed cases and the cases irrelevant to PDR A. baumannii infections are compared in terms of risk factors. 
 
 Result:As a result of the Univariate Analysis, it was found that DM, traumas, CCI>4, steroid use, hospitalization history in the last 3 months, and antibiotic use in the last 3 months were statistically and significantly higher in the PDR A. baumannii Group. Multivariate analysis was used to determine the risk factors with a p value of 0.1 and below by univariate analysis. In this respect, traumas (OR=93.32, p=0.011), steroid use (OR=21.09, p4 olması, steroid kullanımı, son 3 ay hastanede yatış öyküsü ve son 3 ay antibiyotik kullanımı istatiksel anlamlı olarak daha yüksek olduğu bulundu.Univariate analiz ile p değeri 0.1 ve altında saptanan risk faktörleri bağımsız risk faktörlerinin belirlenmesi için multivariate analiz uygulandı.Buna göre travma (OR=93.32, p=0.011), steroid kullanımı (OR=21.09, p

Synergistic Role of Biofilm-Associated Genes and Efflux Pump Genes in Tigecycline Resistance of Acinetobacter baumannii.

BACKGROUND Previous research reported that the resistance mechanism of Acinetobacter baumannii resistance to tigecycline was mainly related to the overexpression of the AdeABC efflux pump system. Biofilm formation is a notable pathogenesis of A. baumannii infections and antibiotic resistance. Our study explores the latent relevance of biofilm-associated genes and efflux pump genes in A. baumannii tigecycline resistance. MATERIAL AND METHODS A total of 78 clinical samples were collected from October 2018 to October 2019. Seventy-two clinically isolated A. baumannii strains were divided into a tigecycline-resistant Acinetobacter baumannii (TR-AN) group and tigecycline-sensitive Acinetobacter baumannii (TS-AN) group by tigecycline minimum inhibitory concentration tests. The biofilm formation of the 2 groups was observed using crystal violet staining. Furthermore, biofilm-related genes and efflux pump genes were analyzed by RT-PCR. RESULTS The biofilm-forming rate of the TR-AN group was 82.2%, and that of the TS-AN group was 14.8%. The biofilm synthesis gene bfs was 91.3% positive in the TR-AN group, significantly higher than in the TS-AN group at the transcription level (P<0.05). The minimum inhibitory concentration of tigecycline was higher in the TR-AN group with biofilm formation than in the TR-AN group without biofilm formation (P<0.05). The efflux pump AdeB gene was 95.2% positive in the TR-AN group with biofilm formation and 38.7% positive in the TR-AN group without biofilm formation. CONCLUSIONS The biofilm formation of A. baumannii may be positively related to tigecycline resistance ability because of the co-expression of the bfs gene and the AdeB efflux pump gene. The enhanced transcription level of bfs and AdeB promotes biofilm formation to improve the resistance of A. baumannii to tigecycline.

Risk factors and cerebrospinal fluid indexes analysis of intracranial infection by Acinetobacter baumannii after neurosurgery

BackgroundIntracranial infection by Acinetobacter baumannii (A. baumannii) after neurosurgery has always been a difficult problem for neurosurgeons. This study analyzed risk factors that discriminated A. baumannii from other bacteria causing intracranial infection after neurosurgery. It also examined the differences in the cerebrospinal fluid (CSF) indexes to explore their value in the early diagnosis of intracranial infection by A. baumannii. MethodsWe retrospectively reviewed ten years (January 2011 to May 2021) of postoperative central nervous system (CNS) infections in the First Hospital of China Medical University. According to the pathogen, CNS infections were divided into A. baumannii group and other species of bacteria group. We collected clinical and laboratory information of patients, and statistical analysis was performed with SPSS 26.0. Risk factors were screened by univariate analysis, and independent risk factors were screened by multiple logistic regression analysis. Finally, CSF-Pro, CSF-Glu, CSF-Cl, CSF-monocytes (%), CSF-multinucleated cells (%) levels, and CSF multinucleated cells%/monocytes% in the different groups were analyzed. ResultsA total of 155 patients were included, 62 cases (40%) of intracranial infection by A. baumannii and 93 cases (60%) by other species of bacteria. The analysis showed that indwelling nasogastric tubes (P＜0.001, OR = 4.231), indwelling peripherally inserted central catheters (PICCs) (P = 0.041, OR = 2.765), and CSF drainage obstruction (P = 0.003, OR = 3.765) were independent risk factors for intracranial infection by A. baumannii after neurosurgery. Indwelling ventriculoperitoneal shunt (VPS) was a protective factor (P = 0.033, OR = 0.22). In addition, compared with other bacterial groups, the A. baumannii group had higher CSF-pro and CSF- multinucleated cells (%) levels and lower CSF-Glu and CSF- monocytes (%) levels, and the difference was statistically significant (P < 0.01). ConclusionsOur results elucidate risk factors and differences in CSF indexes for intracranial infection by A. baumannii after neurosurgery that could be detected and prevented early to reduce mortality.

The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins.

Multidrug-resistant Enterococcus faecium strains are currently a leading cause of difficult-to-treat nosocomial infections. The emerging resistance of enterococci to last-resort antibiotics, such as daptomycin, prompts a search for alternative antimicrobials. Aureocin A53- and enterocin L50-like bacteriocins are potent antimicrobial agents that form daptomycin-like cationic complexes and have a similar cell envelope-targeting mechanism of action, suggesting their potential as next-generation antibiotics. However, to ensure their safe use, the mechanisms of resistance to these bacteriocins and cross-resistance to antibiotics need to be well understood. Here, we investigated the genetic basis of E. faecium's resistance to aureocin A53- and enterocin L50-like bacteriocins and compared it with that to antibiotics. First, we selected spontaneous mutants resistant to the bacteriocin BHT-B and identified adaptive mutations in the liaFSR-liaX genes encoding the LiaFSR stress response regulatory system and the daptomycin-sensing protein LiaX, respectively. We then demonstrated that a gain-of-function mutation in liaR increases the expression of liaFSR, liaXYZ, cell wall remodeling-associated genes, and hypothetical genes involved in protection against various antimicrobials. Finally, we showed that adaptive mutations or overexpression of liaSR or liaR alone results in cross-resistance to other aureocin A53- and enterocin L50-like bacteriocins, as well as antibiotics targeting specific components of the cell envelope (daptomycin, ramoplanin, gramicidin) or ribosomes (kanamycin and gentamicin). Based on the obtained results, we concluded that activation of the LiaFSR-mediated stress response confers resistance to peptide antibiotics and bacteriocins via a cascade of reactions, eventually leading to cell envelope remodeling. IMPORTANCE Pathogenic enterococci carry virulence factors and a considerable resistome, which makes them one of the most serious and steadily increasing causes of hospital epidemiological risks. Accordingly, Enterococcus faecium is classified into a top-priority ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of six highly virulent and multidrug-resistant (MDR) bacterial pathogens for which novel antimicrobial agents need to be developed urgently. Alternative measures, such as the use of bacteriocins, separately or in combination with other antimicrobial agents (e.g., antibiotics), could be a potential solution, especially since several international health agencies recommend and support the development of such interventions. Nevertheless, in order to exploit their efficacy, more basic research on the mechanisms of cell killing and the development of resistance to bacteriocins is needed. The present study fills some of the knowledge gaps regarding the genetic basis of the development of resistance to potent antienterococcal bacteriocins, pointing out the common and divergent features regarding the cross-resistance to antibiotics.

Characterization of Novel Klebsiella Phage PG14 and Its Antibiofilm Efficacy.

The increasing frequency of infections caused by multidrug-resistant Klebsiella pneumoniae demands the development of unconventional therapies. Here, we isolated, characterized, and sequenced a Klebsiella phage PG14 that infects and lyses carbapenem-resistant K. pneumoniae G14. Phage PG14 showed morphology similar to the phages belonging to the family Siphoviridae. The adsorption curve of phage PG14 showed more than 90% adsorption of phages on a host within 12 min. A latent period of 20 min and a burst size of 47 was observed in the one step growth curve. Phage PG14 is stable at a temperature below 30°C and in the pH range of 6 to 8. The PG14 genome showed no putative genes associated with virulence and antibiotic resistance. Additionally, it has shown lysis against 6 out of 13 isolates tested, suggesting the suitability of this phage for therapeutic applications. Phage PG14 showed more than a 7-log cycle reduction in K. pneumoniae planktonic cells after 24 h of treatment at a multiplicity of infection (MOI) of 10. The phage PG14 showed a significant inhibition and disruption of biofilm produced by K. pneumoniae G14. The promising results of this study nominate phage PG14 as a potential candidate for phage therapy. IMPORTANCE Klebsiella pneumoniae is one of the members of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of pathogens and is responsible for nosocomial infections. The global increase of carbapenem-resistant K. pneumoniae has developed a substantial clinical threat because of the dearth of therapeutic choices available. K. pneumoniae is one of the commonly found bacteria responsible for biofilm-related infections. Due to the inherent tolerance of biofilms to antibiotics, there is a growing need to develop alternative strategies to control biofilm-associated infections. This study characterized a novel bacteriophage PG14, which can inhibit and disrupt the K. pneumoniae biofilm. The genome of phage PG14 does not show any putative genes related to antimicrobial resistance or virulence, making it a potential candidate for phage therapy. This study displays the possibility of treating infections caused by multidrug-resistant (MDR) isolates of K. pneumoniae using phage PG14 alone or combined with other therapeutic agents.

First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria.

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

Risk factors and resistance patterns of invasive Acinetobacter Baumannii infection in Children

Objective: To understand the risk factors and antibiotics-resistant patterns of invasive Acinetobacter baumannii infection in Children. Methods: This retrospective study was conducted in 6 tertiary hospitals from January 2016 to December 2018. The basic information, clinical data and the results of antimicrobial susceptibility testing were collected from the 98 pediatric inpatients with Acinetobacter baumannii isolated from blood or cerebrospinal fluid and analyzed. According to the susceptibility of the infected strains to carbapenems, they were divided into carbapenem-sensitive Acinetobacter baumannii (CSAB) group and carbapenem-resistant Acinetobacter baumannii (CRAB) group. According to the possible sources of infection, they were divided into nosocomial infection group and community infection group. Chi-square test or Fisher exact test were used to analyze categorical variables and rank sum test were used to analyze continuous variables. The risk factors of invasive CRAB infection in children were analyzed by Logistic regression. Result: There were 56 males and 42 females in 98 cases. The onset age of patients was 8 (2, 24) months. There were 62 cases (63%) from rural area. A total of 87 cases (89%) were confirmed with bloodstream infection, and 12 cases (12%) confirmed with meningitis (1 case was accompanied with bloodstream infection). In these patients, 66 cases (67%) received invasive medical procedures or surgery, 54 cases (55%) received carbapenems-containing therapy. Twenty-four cases were infected with CRAB, and 74 cases with CSAB. The onset age of cases in CRAB group was lower than that in CSAB group (4 (1, 9) vs. 10 (4, 24) months, Z=-2.16, P=0.031). The proportions of hospitalization in intensive care unit, carbapenem antibiotics using, pneumonia and adverse prognosis in CRAB group were higher than those in CSAB group (6 cases (25%) vs. 4 cases (5%), 18 cases (75%) vs. 36 cases (49%), 17 cases (71%) vs. 17 cases (23%), 6 cases (25%) vs. 4 cases (5%), χ2=5.61, 5.09, 18.32, 5.61, all P<0.05). Seventy-seven cases were nosocomial infection and 21 cases were hospital-acquired infection. The proportion of children hospitalized in high-risk wards for nosocomial infections, length of hospitalization, number of antimicrobial therapy received and duration of antimicrobial therapy were higher in the hospital associated infection group than those in the community acquired infection group (all P<0.05). Logistic regression analysis showed that children from rural area (OR=8.42, 95%CI 1.45-48.88), prior mechanical ventilation (OR=12.62, 95%CI 1.31-121.76), and prior antibiotic therapy (OR=4.90, 95%CI 1.35-17.72) were independent risk factors for CRAB infection. The resistance percentage of CSAB isolates to many classes of antibiotics was <6% except to gentamicin, which was as high as 20% (13/65). All CRAB isolates of resistant to ampicillin-sulbactam (20/20), cefepime (23/23), piperacillin (17/17), meropenem (23/23) and imipenem (24/24) were 100%. The resistance percentage to other antibiotics were up to 42%-96%. Conclusions: Most of invasive Acinetobacter baumannii infection in children in China are hospital-acquired. The outcome of invasive CRAB infection was poorer than that of CSAB infection. The drug resistance rate of CRAB strains isolated is high. Living in rural area, prior invasive mechanical ventilation and prior antibiotic therapy were independent risk factors for invasive CRAB infection. The prevention and control of nosocomial infection and appropriate use of antibiotics to reduce Acinetobacter baumannii infection.

Isolation and characterization of cyromazine degrading Acinetobacter sp. ZX01 from a Chinese ginger cultivated soil.

Cyromazine, a symmetrical triazine insecticide, is used to control dipteran larvae in chicken manure by feeding to the poultry, flies on animals, and leafminers in vegetables. Its extensive use has resulted in the widespread contamination in the environment. In the current study, a cyromazine degrading bacterium (designated strain ZX01) was isolated and characterized from a Chinese ginger cultivated soil by selective enrichment culture method. On the basis of morphological, biochemical characteristics, and 16S rRNA gene sequence, this bacterium showed strong similarity to the Pseudomonadales members and was closely related to the Acinetobacter baumannii group. Spectrophotometric and HPLC analyses revealed that strain ZX01 degraded cyromazine and utilized it as the sole carbon source for its growth. This process hydrolyzes cyromazine to melamine. Strain ZX01 degraded most of the cyromazine in 60h. Besides, its substrate specificity against four symmetrical triazine herbicides, one triazinone herbicide, as well as 10 insecticides and its antibiotic sensitivity towards eight commercial antibiotics were also tested. At the concentration of 100µg/mL for 60h, it could effectively degrade a variety of different pesticides, including atrazine, prometon, simazine, prometryn, enitrothion, diazinon, cypermethrin, and acetamiprid, and the degradation was in the range of 71-87%. In particular, melamine, the main degradation product of cyromazine, was degraded by 47.3%. This microorganism was sensitive to chloramphenicol and tetracycline and intermediate to amoxicillin and trimethoprim. These results highlight that strain ZX01 can be used as a potential biological agent for the remediation of soil, water, or crop contaminated with cyromazine and other symmetrical triazine insecticides.

A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter baumannii Group with Shortened Time-to-Result: rCIM-A.

Carbapenem-resistant Acinetobacter baumannii group organisms (CRAB) are challenging because the choice between targeted, new antibiotic drug options and hygiene measures should be guided by a timely identification of resistance mechanisms. In CRAB, acquired class-D carbapenemases (CHDLs) are active against meropenem and imipenem. If PCR methods are not the first choice, phenotypic methods have to be implemented. While promising, the carbapenemase inactivation method (CIM) using meropenem-hydrolysis is, however, hampered by poor performance or overly long time-to-result. We developed a rapid CIM (rCIM-A) with good performance using ertapenem, imipenem, and meropenem disks, 2-h permeabilization and incubation with the test strain in trypticase soy broth, and a read-out of residual carbapenem activity after 6 h, and optionally after 16–18 h. Using clinical isolates and type-strains of Acinetobacter (n = 67) not harboring carbapenemases (n = 28) or harboring acquired carbapenemases (n = 39), the sensitivity of detection was 97.4% with the imipenem disk after 6 h at a specificity of 92.9%. If the inhibition zone around the ertapenem disk at 6 h was 6 or ≤26 mm at 16–18 h, or ≤25.5 mm for meropenem, the specificity was 100%. Because of the high negative predictive value, the rCIM-A seems particularly appropriate in areas of lower CRAB-frequency.

The Three Main SCFAs Inhibit the Inflammatory Response of A549 Cells Induced by &amp;lt;i&amp;gt;Acinetobacter baumannii&amp;lt;/i&amp;gt;

Objective: The objective is to explore the mechanism of inhibitory effect of three main SCFAs (acetate, propionate and butyrate) on inflammatory response of A549 cells. Methods: Human lung adenocarcinoma cells (A549 cells) were cultured, and were divided into normal control group (NC group), A. baumannii infection group (A. baumannii group), NF-κB inhibitor group (JSH group), A. baumannii infection + sodium acetate group (NaAc group), A. baumannii infection + sodium propionate group (NaPc group) and A. baumannii infection + sodium butyrate group (NaB group). Real-time quantitative PCR was used to detect the mRNA expression of NLRP3, Caspase-1, IL-1β, IL-6, and TGF-β in A549 cells. Western blotting assay was used to determine the expression of autophagy and “pyroptosis” related proteins of NRLP3, cleaved-Caspase-1 (P20), GSDMD (P30), LC-3 and Beclin-1. At the same time, the expression of NF-κB p65 protein in nucleus and cytoplasm of A549 cells was detected. The level of reactive oxygen species in A549 cells was detected by flow cytometry. Results: Compared with A. baumannii group, the mRNA expression of NLRP3, IL-1β and IL-6 in NaAc group, NaPc group and NaB group decreased significantly, the mRNA expression of Caspase-1 in NaPc group and NaB group decreased significantly, only the mRNA expression of TGF-β in NaB group increased significantly; LC3-II expression increased significantly in NaPc group and NaB group, only Beclin-1 expression increased and GSDMD (p30) expression decreased significantly in NaB group. All three kinds of SCFAs could significantly inhibit the expression of cleaved-Caspase-1 (p20) after A. baumannii infection, but there was no significant change in the protein expression of NLRP3. Compared with NC group, the production of reactive oxygen species in A. baumannii group increased significantly at 3 h after A. baumannii infection. Compared with A. baumannii group, NaB could significantly suppress the production of reactive oxygen species induced by A. baumannii. Compared with A. baumannii group, the expression of NF-κB p65 in nucleus was significantly decreased and the expression of NF-κB p65 in cytoplasm was significantly increased after 24 h pre-incubation with NaB, NaPc and NaAc, respectively. Conclusion: A. baumannii can induce inflammatory injury of pulmonary epithelial cells, and the three major SCFAs can inhibit the activation of NLRP3 inflammasome and the release of pro-inflammatory factors through NF-κB/ROS/NLRP3 pathway, which provides a new way for clinical prevention of severe inflammatory injury caused by A. baumannii infection.

Multidrug-Resistant Acinetobacter baumannii Genetic Characterization and Spread in Lithuania in 2014, 2016, and 2018.

Bacterial resistance to antimicrobial agents plays an important role in the treatment of bacterial infections in healthcare institutions. The spread of multidrug-resistant bacteria can occur during inter- and intra-hospital transmissions among patients and hospital personnel. For this reason, more studies must be conducted to understand how resistance occurs in bacteria and how it moves between hospitals by comparing data from different years and looking out for any patterns that might emerge. Multidrug-resistant (MDR) Acinetobacter spp. was studied at 14 healthcare institutions in Lithuania during 2014, 2016, and 2018 using samples from human bloodstream infections. In total, 194 isolates were collected and identified using MALDI-TOF and VITEK2 analyzers as Acinetobacter baumannii group bacteria. After that, the isolates were analyzed for the presence of different resistance genes (20 genes were analyzed) and characterized by using the Rep-PCR and MLVA (multiple-locus variable-number tandem repeat analysis) genotyping methods. The results of the study showed the relatedness of the different Acinetobacter spp. isolates and a possible circulation of resistance genes or profiles during the different years of the study. This study provides essential information, such as variability and diversity of resistance genes, genetic profiling, and clustering of isolates, to better understand the antimicrobial resistance patterns of Acinetobacter spp. These results can be used to strengthen the control of multidrug-resistant infections in healthcare institutions and to prevent potential outbreaks of this pathogen in the future.

Initial white blood cell count and revised Baux score predict subsequent bloodstream infection in burn patients: A retrospective analysis of severe burn patients from the Formosa color dust explosion of 2015

Infections are the most common complications among hospitalized severe burn patients. However, limited literature reports early effective predictors of bloodstream infections (BSI) among burn patients. This study aimed to identify cost-effective biomarkers and valuable clinical scoring systems in the emergency department (ED) for the prediction of subsequent BSI in mass burn casualties. In 2015, a flammable cornstarch-based powder explosion resulted in 499 burn casualties in Taiwan. A total of 35 patients were admitted at Taipei Veterans General Hospital. These severe burn patients (median total body surface area [TBSA] 54%) were young and previously healthy. We assessed the potential of various parameters to predict subsequent BSI, including initial laboratory tests performed at the ED, TBSA, and multiple scoring systems. Fourteen patients (40.0%) had subsequent BSI. The most common causative pathogen was the Acinetobacter baumannii (Ab) group, mostly carbapenem resistant and associated with a poor outcome. The area under the receiver operating characteristic curve revealed that the revised Baux score, TBSA, and initial white blood cell count had excellent discrimination ability in predicting subsequent BSI (0.898, 0.889, and 0.821, respectively). The rate of subsequent BSI differed significantly at the cut-off points of revised Baux score >76, TBSA >55%, and WBC count >16,200/mm3. The initial WBC count at the ED, TBSA, and revised Baux score were good and cost-effective biomarkers for predicting subsequent BSI after burn injuries.

Clinical and molecular characterization of Acinetobacter seifertii in Taiwan.

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. 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. 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. 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.

Acinetobacter: An Enemy after Head and Neck Cancer Operations with Microvascular Free Flap Reconstruction?

Background: Patients after head and neck cancer reconstructive surgical procedures are predisposed to have post-operative surgical site infections (SSI) develop. They are very often caused by multi-drug resistant strains, including Acinetobacter baumannii as the most common one. Methods: The aim of the study was to determine important risk factors contributing to SSI of A. baumannii origin. The analysis included 134 head and neck cancer patients after salvage operations with microvascular free flap reconstruction. The A. baumannii was cultured in 27 of all 48 infected patients. Results: The following risk factors were significantly associated with A. baumannii infection: re-hospitalization before reconstructive operation (p = 0.00011), massive blood loss (p = 0.00277), and need of revision surgical procedure (p = 0.00419). Of patients with A. baumannii infection, 48% were hospitalized in a general intensive care unit (ICU) after operation that, together with prolonged intubation, constituted a strong risk factor of that infection (p = 0.01077). Mean time of hospital stay was significantly longer in the A. baumannii group (58 days vs. 35 days; p = 0.02697). Conclusions: Our analysis identified a subset of head and neck cancer patients after salvage operation with microvascular free flap reconstruction who are at high risk of A. baumannii infection developing. Previously hospitalized patients with extensive blood loss and need of surgical revision necessitate increased monitoring for the development of this complication. Mechanical ventilation and hospital stay in an ICU should be shortened maximally or avoided in that challenging group of patients. Early recognition of patients at high risk remains a key point to prevent or limit the spread of A. baumannii infections.

Method for phenotypic identification of &lt;i&gt;Acinetobacter nosocomialis&lt;/i&gt; bacteria

The study's purpose was the developing of the method for Acinetobacter nosocomialis bacteria identification by the totality of phenotypic characteristics of the Acinetobacter baumannii (Ab) group based on the urease activity trait.The research's objects were clinical strains of the Ab group, isolated in 2018—2019 in the bacteriological laboratory of Kirov Military Medical Academy (St. Petersburg), of which were A. baumannii (n = 85), A. nosocomialis (n = 12), A. pittii (n = 10). In addition, 9 strains of A. nosocomialis were isolated from water samples from the Neva River and 2 strains of A. calcoaceticus from the cyanobacterial mats. The belonging of the strains to Ab group was determined by the combination of the metabolic and physiological characteristics of the taxonomic tests for this group. The species identification of the studied strains was determined by the matrix-associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The urease activity of bacteria was determined using the microvolume method. We used a medium with urea in the following composition (g/l): Na2HPO4 — 1.1; KH2PO4 — 1.1; NaCI — 5.0; 40% alkaline solution phenol red — 5 ml; urea — 10—15; distilled water — 1 l. The ingredients, without urea, were dissolved in distilled water, dispensed to vials, sterilized for 20 minutes at 121°C, urea was added in the calculated amount. The medium with urea was added in 0.1 ml to wells of the plate, then inoculated the daily agar culture of the studied and control strains by full loop (2 mm in diameter) and incubated under aerobic conditions at 37°C. The reaction results for a rapid urease activity were determined after 3 hours by a change in the initial color of the medium. The reactions were accounted after 7—24 hours to detect activity urease. It was found that 100% of A. nosocomialis strains and 18.82% of A. baumannii strains had urease activity. At the same time, high activity urease was found only in one strain of A. baumannii (1.17%) and in all strains of A. nosocomialis. Therefore, the presence of high urease activity is of taxonomic importance for the species A. nosocomialis as the marker of distinguishing this species from other bacteria species of the Ab group. The sensitivity and specificity of the identifying A. nosocomialis strains by suggested approach compared to the studied strains identification by MALDI-TOF MS were 100 and 98.83% (x2 = 103.2; p 0.0001), respectively.

In vitro activity of cefiderocol against aerobic Gram-negative bacterial pathogens from Germany

Objectives: Cefiderocol (CID), also known as S-649266, a novel siderophore cephalosporin, possesses potent activity against multidrug-resistant aerobic Gram-negative bacteria (GNB). This study aimed to determine the in vitro activity of CID against two different sets of GNB: i) a random sample of 213 clinical isolates, including 17 extended-spectrum beta-lactamase (ESBL) producers, obtained from intensive care unit patients with nosocomial infections collected during a multicentre surveillance study (set I); and ii) a group of 59 challenge GNB producing various types of carbapenemases (CP; set II).Methods: Minimum inhibitory concentrations (MICs) were determined using the microdilution method according to the standard ISO 20776-1. Iron-depleted medium was used for testing CID.Results: CID inhibited 97.2% of set I isolates at the EUCAST susceptibility breakpoint of ≤ 2 mg/L. The concentrations of CID inhibiting 50% and 90% (MIC50/90) of the Enterobacterales isolates (n = 146) were 0.12/1.0 mg/L, with ESBL-positive isolates tending to exhibit higher MICs than ESBL-negative isolates to CID. MIC50/90 values of CID for isolates of the Acinetobacter baumannii group (n = 13) and Pseudomonas aeruginosa (n = 54) were 0.06/0.12 mg/L and 0.12/0.5 mg/L, respectively. Further, CID inhibited 88.1% of set II CP-producing isolates at ≤ 2 mg/L. All seven class D CP-producing Acinetobacter baumannii were inhibited at ≤ 0.25 mg/L. MIC50/90 values for CP-producing Enterobacterales (n = 30) and Pseudomonas aeruginosa (n = 22) were 1/4 mg/L and 0.5/2 mg/L, respectively.Conclusion: CID showed potent activity against Acinetobacter baumannii, Enterobacterales and Pseudomonas aeruginosa, including CP-producing isolates. Overall, CID inhibited 259 of 272 (95.2%) GNB at ≤ 2 mg/L.

Evaluation of the performance of Acuitas® Resistome Test and the Acuitas Lighthouse® software for the detection of β-lactamase-producing microorganisms

ObjectivesGiven the international spread of multidrug-resistant Gram-negative bacteria the need for prompt and precise characterization of underlying resistance traits has evolved into the cornerstone of infection control strategies. Novel commercial molecular tests enable rapid simultaneous testing for multiple resistance genes. We aimed to evaluate the performance of OpGen’s Acuitas® Resistome Test and the Acuitas Lighthouse® software. MethodsThe test is tailored towards detecting 46 β-lactamase genes (SHV and TEM variants associated with wild-type penicillin resistance, extended-spectrum β-lactamases [ESBLs], acquired AmpCs and carbapenemases) via a microfluidic polymerase chain reaction (PCR) array. In total 118 isolates part of the collection of the Bacteriology Laboratory of the Hellenic Pasteur Institute, specifically 96 enterobacterial isolates and 21 Acinetobacter baumannii, of divergent origins, with previously characterized β-lactamase content, were tested. ResultsIn the enterobacterial group all 69 carbapenemase genes of the KPC, VIM, NDM and OXA-48 types were correctly identified (sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] of 100%). Non-ESBL SHV enzymes, ESBLs (CTX-M, GES, VEB types) and acquired AmpC enzymes were also correctly characterized. Of the 35 SHV-ESBLs harboured, correct identification was possible in 32/35 isolates, with overall sensitivity, specificity, PPV and NPV for the Klebsiella pneumoniae group of 89.29%, 100%, 100% and 91.18%, respectively. For the A. baumannii group the test exhibited an overall sensitivity for carbapenemase detection of 96.55% and 100% PPV. ConclusionsThe OpGen Acuitas Resistome Test is an efficient molecular tool that can identify resistance threats in health care institutions with high diagnostic accuracy and be integrated into targeted surveillance protocols.