Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

[Analysis of the virulence and genetic differences of carbapenem-resistant Acinetobacter baumannii epidemic clones].

Objective: To evaluate the virulence levels of carbapenem-resistant Acinetobacter baumannii ST191, ST195, and ST208, and to analyze the differences in virulence factors among these epidemic clones. Methods: The study involved the genomic sequencing of 233 Acinetobacter baumannii strains that were isolated from the Fifth Medical Center of the Chinese People's Liberation Army General Hospital (North Hospital) between 2011 and 2019. The genomic data was cross-referenced with the Virulence Factor Database (VFDB) to examine the presence of virulence genes in the strains. Furthermore, a Galleria mellonella infection survival model was used to evaluate the virulence levels of the strains, and the association between virulence levels and virulence genes was analyzed. Results: The study included 38 strains of the ST191 clone, 104 strains of the ST195 clone, and 91 strains of the ST208 clone. In the Galleria mellonella infection survival experiment, the average mortality rate for ST191 was 23.0%, with 3 (7.9%) highly virulent strains. For ST195, the average mortality rate was 53.0%, with 34 (32.7%) highly virulent strains. For ST208, the average mortality rate was 47.0%, with 20 (21.9%) highly virulent strains. There was a significant statistical difference in mortality rates between ST191 and ST195 (χ2=13.9, P<0.001) as well as between ST191 and ST208 (χ2=15.2, P<0.001). A comparison of the strains with the VFDB revealed significant differences in the virulence genes carried by the clones. Specifically, the type Ⅵ secretion system-related genes (clpV/tssH, hcp/tssD, tagX, tssA, tssB, tssC, tssE, tssF, tssG, tssK, ssL, tssM) and the sugar transferase gene ACICU_RS00475 were found to be universally absent in ST191 strains (0%) while being prevalent in ST195 (100.0%) and ST208 (>82.0%) strains. Statistical analysis revealed an association between the mortality rate of the clones and the presence of virulence genes(clpV/tssH P<0.001, hcp/tssD P=0.001, tagX P<0.001, tssA P<0.001, tssB P=0.001, tssC P=0.001, tssE P=0.001, tssF P=0.001, tssG P<0.001, tssK P<0.001, tssL P<0.001, tssM P=0.001, ACICU_RS00475 P=0.001). Conclusion: Among the carbapenem-resistant epidemic clones of Acinetobacter baumannii, the ST191 clone shows lower mortality rates in Galleria mellonella, possibly because of the lack of type Ⅵ secretion system and sugar transferase genes.

Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

Molecular epidemiology and molecular typing methods of Acinetobacter baumannii: An updated review.

The aim of this study was to go through the molecular methods used for typing of carbapenem-resistant Acientobacter baumannii (CRAB) isolates for investigating the molecular epidemiology all over the world. Multiple typing techniques are required to understand the source and nature of outbreaks caused by Acientobacter baumannii (A. baumannii) and acquired resistance to antimicrobials. Nowadays, there is gradual shift from traditional typing methods to modern molecular methods to study molecular epidemiology and infection control. Molecular typing of A. baumannii strains has been revolutionized significantly in the last 2 decades. A few sequencing-based techniques have been proven as a breakthrough and opened new prospects, which have not been achieved by the traditional methods. In this review, discussed different pre-existing and recently used typing methods to explore the molecular epidemiology of A. baumannii pertaining in context with human infections.

Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Lactate metabolism promotes in vivo fitness during Acinetobacter baumannii infection.

Acinetobacter baumannii is one of the most prevalent causes of nosocomial infections worldwide. However, a paucity of information exists regarding the connection between metabolic capacity and in vivo bacterial fitness. Elevated lactate is a key marker of severe sepsis. We have previously shown that the putative A. baumannii lactate permease gene, lldP, is upregulated during in vivo infection. Here, we confirm that lldP expression is upregulated in three A. baumannii strains during a mammalian systemic infection. Utilising a transposon mutant disrupted for lldP in the contemporary clinical strain AB5075-UW, and a complemented strain, we confirmed its role in the in vitro utilisation of l-(+)-lactate. Furthermore, disruption of the lactate metabolism pathway resulted in reduced bacterial fitness during an in vivo systemic murine competition assay. The disruption of lldP had no impact on the susceptibility of this strain to complement mediated killing by healthy human serum. However, growth in biologically relevant concentrations of lactate observed during severe sepsis, led to bacterial tolerance to killing by healthy human blood, a phenotype that was abolished in the lldP mutant. This study highlights the importance of the lactate metabolism pathway for survival and growth of A. baumannii during infection.

Phage-Bacterial Interaction Alters Phenotypes Associated with Virulence in Acinetobacter baumannii.

Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.

Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Exploring the evolutionary and pathogenic role of Acinetobacter baumannii biofilm-associated protein (Bap) through in silico structural modeling.

Acinetobacter species encode for extracellularly secreted Biofilm-associated protein (Bap), a multi-domain protein with variable molecular weights reaching several hundred kilodaltons. Bap is crucial for the development of multi-dimensional structures of mature biofilms. In our investigation, we analyzed 7338 sequences of A. baumannii from the NCBI database and found that Bap or Bap-like protein (BLP) was present in 6422 (87.52%) isolates. Further classification revealed that 12.12% carried Type-1 Bap, 68.44% had Type-2, 6.91% had Type-3, 0.05% had Type-6 or SDF-Type, and 12.51% lacked Bap or BLP. The majority of isolates with Type-1, Type-2, and Type-3 Bap belonged to ST1, ST2, and ST25, respectively. Phylogenetic analysis suggested that Type-1 Bap is the most ancient, while Type-3 and SDF-Type have evolved recently. Studying the interaction of predicted Bap structures with human CEACAM-1 and PIgR showed that Bap with its BIg13 and BIg6 domains interact with the N-terminal domain of CEACAM-1, involving Arg43 and Glu40, involved in CEACAM-1 dimerization. Also, we found that recently evolved Type-3 and SDF-Type Bap showed greater interaction with CEACAM-1 and PIgR. It can be asserted that the evolution of Bap has conferred enhanced virulence characteristics to A. baumannii with increased interaction with CEACAM-1 and PIgR. Using in silico approaches, this study explores the evolutionary, physicochemical, and structural features of A. baumannii Bap and unravels its crucial role in mediating interaction with human CEACAM-1 and PIgR through detailed structure modelling. These findings advance our understanding of A. baumannii Bap and highlight its role in pathogenesis.

Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Seasonal meropenem resistance in Acinetobacter baumannii and influence of temperature-driven adaptation.

BACKGROUND: Recognition of seasonal trends in bacterial infection and drug resistance rates may enhance diagnosis, direct therapeutic strategies, and inform preventive measures. Limited data exist on the seasonal variability of Acinetobacter baumannii. We investigated the seasonality of A. baumannii, the correlation between temperature and meropenem resistance, and the impact of temperature on this bacterium. RESULTS: Meropenem resistance rates increased with lower temperatures, peaking in winter/colder months. Nonresistant strain detection exhibited temperature-dependent seasonality, rising in summer/warmer months and declining in winter/colder months. In contrast, resistant strains showed no seasonality. Variations in meropenem-resistant and nonresistant bacterial resilience to temperature changes were observed. Nonresistant strains displayed growth advantages at temperatures ≥ 25 °C, whereas meropenem-resistant A. baumannii with ß-lactamase OXA-23 exhibited greater resistance to low-temperature (4 °C) stress. Furthermore, at 4 °C, A. baumannii upregulated carbapenem resistance-related genes (adeJ, oxa-51, and oxa-23) and increased meropenem stress tolerance. CONCLUSIONS: Meropenem resistance rates in A. baumannii display seasonality and are negatively correlated with local temperature, with rates peaking in winter, possibly linked to the differential adaptation of resistant and nonresistant isolates to temperature fluctuations. Furthermore, due to significant resistance rate variations between quarters, compiling monthly or quarterly reports might enhance comprehension of antibiotic resistance trends. Consequently, this could assist in formulating strategies to control and prevent resistance within healthcare facilities.

Molecular characterization of carbapenemase and extended spectrum beta-lactamase producing Acinetobacter baumannii isolates causing surgical site infections in Ethiopia.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that can cause a variety of nosocomial infections in humans. This study aimed to molecularly characterize extended-spectrum beta-lactamase (ESBL) producing and carbapenem-resistant Acinetobacter species isolated from surgical site infections (SSI). METHODS: A multicentre cross-sectional study was performed among SSI patients at four hospitals located in Northern, Southern, Southwest, and Central parts of Ethiopia. The isolates were identified by microbiological methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was determined using disk diffusion. The presence of phenotypic ESBL and carbapenemase production was detected by employing standard microbiological tests, including combined disk diffusion (CDT). ESBL and carbapenem resistance determinants genes were studied by polymerase chain reaction (PCR) and sequencing. RESULTS: A total of 8.7% Acinetobacter species were identified from 493 culture-positive isolates out of 752 SSI wounds. The species identified by MALDI-TOF MS were 88.4% A. baumannii, 4.7% Acinetobacter pittii, 4.7% Acinetobacter soli, and 2.3% Acinetobacter lactucae. Of all isolates 93% were positive for ESBL enzymes according to the CDT. Using whole genome sequencing 62.8% of the A. baumannii harbored one or more beta-lactamase genes, and 46.5% harbored one or more carbapenemase producing genes. The distribution of beta-lactamases among Acinetobacter species by hospitals was 53.8%, 64.3%, 75%, and 75% at JUSH, TASH, DTCSH, and HUCSH respectively. Among ESBL genes, blaCTX-M alleles were detected in 21.4% of isolates; of these 83.3% were blaCTX-M-15. The predominant carbapenemase gene of blaOXA type was detected in 24 carbapenem-resistant A. baumannii followed by blaNDM alleles carried in 12 A. baumannii with blaNDM-1 as the most common. CONCLUSIONS: The frequency of Acinetobacter species that produce metallobetalactamases (MBLs) and ESBLs that were found in this study is extremely scary and calls for strict infection prevention and control procedures in health facilities helps to set effective antibiotics stewardship.

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

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

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

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

Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020-2022 retrospective study.

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020-2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles.

Characteristics of the Genetic Spread of Carbapenem-Resistant Acinetobacter baumannii in a Tertiary Greek Hospital.

Acinetobacter baumannii (Ab) has increasingly been identified as a cause of hospital-acquired infections and epidemics. The rise of carbapenem-resistant Acinetobacter baumannii (CRAB) poses significant challenges in treatment. Nosocomial outbreaks linked to CRAΒ A. baumannii strains have been reported worldwide, including in Greece. This study aimed to analyze the molecular epidemiology trends of multidrug-resistant A. baumannii isolates in a tertiary hospital in Athens, Greece. A total of 43 clinical isolates of extensively drug-resistant (XDRAB), pan-drug-resistant (PDRAB), and CRAB were collected from patients suffering from blood infection, hospitalized between 2016 and 2020 at the internal medicine clinics and the ICU. A.baumannii isolates underwent testing for Ambler class B and D carbapenemases and the detection of ISAba1, and were typed, initially, using pulsed-field gel electrophoresis, and, subsequently, using sequence-based typing and multiplex PCR to determine European Clone lineages. The blaOXA-23 gene accompanied by ISAba1 was prevalent in nearly all A. baumannii isolates, except for one carrying blaOXA-58. The intrinsic blaOXA-51-like gene was found in all isolates. No Ambler class B carbapenemases (VIM, NDM) were detected. Isolates were grouped into four PF-clusters and no one-cluster spread was documented, consistent with the absence of outbreak. The study indicated that XDR/PDR-CRAB isolates predominantly produce OXA-23 carbapenemase and belong to European Clone II. Further research is needed to understand the distribution of resistant bacteria and develop effective prevention and control strategies.

[Comparison of Three Different Methods in Investigating the Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates]. / Karbapeneme Dirençli Acinetobacter baumannii Klinik Izolatlarinin Moleküler Epidemiyoloji Açisindan Arastirilmasinda Üç Farkli Yöntemin Karsilastirilmasi.

The aim of the study was to evaluate the relationship between carbapenem-resistant Acinetobacter baumannii isolates carrying oxacillinase-type carbapenemase genes with "international high-risk clones" (IC I, II, and III) by different molecular epidemiological methods and to statistically compare the concordance and discrimination power of the methods. Carbapenem-resistant and moderately susceptible A.baumannii isolates from non-repeating blood cultures of 72 patients were included in the study. The presence of "blaOXA-23 , blaOXA-24 , blaOXA-51 ve blaOXA-58 " genes within OXA-type carbapenemases was detected by polymerase chain reaction (PCR) method and confirmed by DNA sequence analysis. Pulsed f ield gel electrophoresis (PFGE), multilocus sequence typing (MLST) and matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS) analyses were performed to evaluate the clonal relations of IC I, II and III clones together with clinical isolates. In the statistical comparison of the methods, discrimination power was evaluated by Simpson index of diversity (SID) and concordance by "Wallace coefficient". All of the isolates were found to carry blaOXA-23 and blaOXA-51 genes. As a result of the bioinformatic analysis of the four isolates selected for sequence analysis; blaOXA-23 and blaOXA-51 genes were detected in the selected isolates, and the analysis of two isolates carrying blaOXA-51 gene showed 99% similarity with blaOXA-92 gene. The isolates were clustered into five pulsotypes (A, B, C, D and E) according to ≥ 85% similarity coefficient by PFGE. The isolates and RUH 875, RUH 134, LUH 5875 strains belonging to high-risk clones ICI, ICII and ICIII, respectively, were divided into five main groups [A (n= 58), B (n= 8), C (n= 4), D (n= 4) and E (n= 1)] and 10 subgroups (A1, A2, A4, A5, A6, A9, B1, B4, C3, D1) by PFGE. IC clone III (E1) and seven strains showed singleton PFGE profiles (A3, A7, A8, B2, B3, C1, C2). ICII was found in A5 subtype, ICI in C1 subtype and ICIII in E1 subtype. By PFGE subtype groups, 18 pulsotypes were determined and ST1, ST2, ST81, ST157 and ST604 sequence types were found in 20 isolates randomly selected from pulsotypes according to MLST Pasteur scheme (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB). Principal component analysis (PCA) of the spectra of 72 A. baumannii isolates and ICI, ICII and ICIII clones was performed by MALDI-TOF MS. In PCA analysis, the cluster distance level was defined as 1.5 and the isolates were divided into three clusters. IC clone I, II and III together with 70 clinical isolates were grouped in one cluster, while two clinical isolates (AB083 and AB0115) formed singleton clusters. There was no significant agreement between MALDI-TOF MS; MLST and PFGE data according to Wallace coefficient. It was found that PFGE method gave significant results in terms of discrimination power with SID coefficient, MALDI-TOF MS PCA analysis had the lowest discrimination power value, and the Wallace coefficient result of PFGE and MLST was concordant. In conclusion, MALDI-TOF MS may not function as a gold standard method like PFGE and MLST for epidemiological analysis in A.baumannii species and the epidemiological typing protocols used for MALDI-TOF MS need to be improved and developed.

Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

AIMS: Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, ß-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. METHODS AND RESULTS: The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. CONCLUSIONS: This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

Discovery of Streptomyces species CS-62, a novel producer of the Acinetobacter baumannii selective antibiotic factumycin.

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation.