Clinical Isolates Of Acinetobacter Research Articles

Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

713. Prevalence of Microbicide Resistance Genes in Clinical Isolates of Acinetobacter baumannii

Abstract Background The persistence of Acinetobacter on different surfaces and the continuous exposure of bacteria to disinfectants such as quaternary ammonium compounds (QACs) in the healthcare environment have led to microbicide tolerance. Acinetobacter baumannii have also rapidly acquired carbapenem resistance. Here we determined the prevalence of microbicide resistance genes in carbapenem susceptible and resistant clinical isolates of Acinetobacter. Methods All clinical isolates were collected from two separate hospitals in Detroit between 2017-2021. Following DNA isolation using Qiagen kit, Nextera Flex kit was used for library preparation of Acinetobacter clinical isolates. Whole genome sequencing (WGS) was performed via Illumina NextSeq 550 using the prepared DNA library. The FASTQ files from the sequencing run were then subjected to bioinformatic analysis with Bionumerics software v7.6. ResFinder was used for determination of presence or absence of QAC resistant genes and results were tabulated by Carbapenem-Resistant Acinetobacter baumannii (CRAb) or Carbapenem-Susceptible Acinetobacter baumannii (CSAb) with or without the QAC resistant genes. Results Out of the 139 Acinetobacter patient isolates, 70/139 (50.4%) were CRAb containing carbapenemase blaOXA-23 and 69/139 (49.6%) belonged to CSAb (Table 1). Figure 1. shows the prevalence of antimicrobial resistance (AMR) genes in CRAb and CSAb isolates with qacE genes. For CRAb isolates, 56% had qacE genes while 39% of CSAb had qacE genes. About 42% of CRAb isolates were multi-drug resistant (MDR), indicating a prevalence of qacE genes among the MDR isolates. Table 2 shows the presence of qacE genes by sequence type (ST). In the ST2 (Pasteur) and ST406 (Pasteur) strains, qacE gene was mainly present. Interestingly, most of the Acinetobacter isolates that belong to ST2 (ST195 and ST281, Oxford) are CRAb with qacE gene.Figure 1:Different AMR genes and qacE in both CRAb and CSAb isolates Conclusion Clinical isolates in our study had an even distribution of both CRAb and CSAb. The presence of qacE gene was more prevalent in the CRAb isolates compared to the CSAb isolates. While different STs harbored the qacE gene, the prevalence of qacE gene was highest in ST2 among all isolates. Future studies are needed to determine the reduced susceptibility to other commonly used hospital disinfectants. Disclosures Keith S. Kaye, MD, MPH, Abbvie: Advisor/Consultant|Abbvie: Honoraria|Entasis: Advisor/Consultant|Entasis: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|Merck: Advisor/Consultant|Merck: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|VenatoRx: Advisor/Consultant|VenatoRx: Honoraria

Colistin and carbapenem resistance among Pseudomonas and Acinetobacter clinical isolates in Menoufia University Hospitals

Colistin and carbapenem resistance among Pseudomonas and Acinetobacter clinical isolates in Menoufia University Hospitals

Heteroduplex Typing of Clinical Isolates of Acinetobacter from Intensive Care Units in a Tertiary Health Care Centre and their Antibiogram

Background Acinetobacter is an important hospital pathogen whose role is implicated in various disease conditions especially in patients confined to hospital intensive care units. Differentiation of isolates is essential to identify the source and to understand the spread which in turn helps to prevent infections. HDA being a sensitive reliable and rapid method a large number of isolates can be typed using this method. Therefore we have adapted this technique to differentiate between clinical isolates of Acinetobacter. Aims Heteroduplex analysis to type clinical isolates of Acinetobacter. Methodology 570 clinical isolates of Acinetobacter from different ICUs were analysed. Antibiogram was performed using VITEK system. DNA extraction was done by phenol-chloroform method. Acinetobacter PCR was performed by targeting 397 bp hyper variable region of rpoB gene. The Heteroduplex assay was performed by mixing the amplicons of test strains and that of in-house standard strain. The formation of Heteroduplexes was determined by polyacrylamide gel electrophoresis. Results Out of 570 clinical isolates 212 37.19 isolates were typed into one of the 25 HD types. Remaining 358 62.81 isolates did not form Heteroduplex with in-house standard. No significant variation was seen in the Antibiogram of different Heteroduplex types. Conclusion Heteroduplex analysis provides a simple and accurate method to analyse the strain diversity of Acinetobacter isolates. Epidemiological typing and source tracing of the pathogens cab be done using HD typing. The technique has potential to be applied for other organisms. Various gene targets in the same pathogen may be explored for better precision.

Clonal diversity of Acinetobacter clinical isolates producing NDM-type carbapenemase in Cuba, 2013–19

ObjectivesOur study aimed to elucidate the clonal diversity of carbapenem-resistant Acinetobacter clinical isolates producing NDM-type carbapenemase collected through national surveillance in Cuba during a 7-year period (2013−19). MethodsA total of 199 isolates of Acinetobacter spp. from 37 hospitals in 12 provinces were genetically analyzed for their species, carbapenemase genes and genotypes. Sequence type (ST) and OXA-51-like gene type were determined for blaNDM-positive isolates. ResultsMost isolates (95%) were identified as species of Acinetobacter calcoaceticus-baumannii complex, with A. baumannii being the majority. Acquired carbapenemase genes were assigned to blaOXA or blaNDM type; the most commonly detected gene was blaOXA-23-like (49%), followed by blaOXA-24-like (20%) and blaNDM (15%). Twenty-nine blaNDM-positive isolates (22 A. baumannii, 2 A. pittii, 2 A. johnsonii, 3 other species) were differentiated into 19 STs, including the most common, ST23. Though NDM genes were mostly typed as blaNDM-1, a novel blaNDM-42 was identified in an ST79 A. baumannii isolate. blaOXA-51-like genes of NDM-positive A. baumannii were discriminated into 10 OXA types, including 2 novel ones. ConclusionsOur study indicated the spread of blaNDM to various clones of A. baumannii and other Acinetobacter spp. in Cuba.

Clinical Isolates of Acinetobacter spp. Are Highly Serum Resistant Despite Efficient Recognition by the Complement System.

Gram-negative bacteria from the genus Acinetobacter are responsible for life-threating hospital-related infections such as pneumonia, septicemia, and meningitis, especially in immunocompromised patients. Worryingly, Acinetobacter have become multi- and extensively drug resistant (MDR/XDR) over the last few decades. The complement system is the first line of defense against microbes, thus it is highly important to increase our understanding of evasion mechanisms used by Acinetobacter spp. Here, we studied clinical isolates of Acinetobacter spp. (n=50), aiming to characterize their recognition by the complement system. Most isolates tested survived 1 h incubation in 30% serum, and only 8 isolates had a lower survival rate, yet none of those isolates were fully killed. Intriguingly, four isolates survived in human whole blood containing all cell component. Their survival was, however, significantly reduced. Flow cytometry analyses revealed that most of the isolates were detected by human IgG and IgM. Interestingly, we could not detect any significant concentration of deposited C1q, despite observing C4b deposition that was abolished in C1q-deficient serum, indicating transient binding of C1q to bacteria. Moreover, several isolates were recognized by MBL, with C4b deposition abolished in MBL-deficient serum. C3b was deposited on most isolates, but this was not, however, seen with respect to C5b and formation of the membrane attack complex (MAC), indicating that many isolates could avoid complement-mediated lysis. India ink staining showed that isolates were capsulated, and capsule thickness varied significantly between isolates. Studies performed on a wild-type strain and capsule mutant strains, demonstrated that the production of a capsular polysaccharide is one mechanism that mediates resistance to complement-mediated bactericidal activity by preventing MAC deposition and lysis. Our data showed that most clinical Acinetobacter spp. isolates are highly serum resistant despite being efficiently recognized by the complement system.

Determination of In vitro Antimicrobial activity of copper on the clinical isolates of Acinetobacter spp

Background: Acinetobacter spp., emerging pathogens equipped with the competence to establish multitudinous severe infections in immunocompromised hosts, are grievous threats to human health. To tackle the enormous burden of disease caused by Acinetobacter spp., the headlong discovery and the advancement of novel therapies are of the essence at this juncture. The present study attempted to determine the antimicrobial effects of copper on the clinical isolates of Acinetobacter spp. (Iso-03 and Iso-04). Methods: The potential deployment of copper-based antibacterial strategies against Acinetobacter spp. was assessed by exposing the isolates to the increasing concentrations of CuSO4 (from 2.5 to 1.5 mM) in liquid culture (M9 minimal medium) for 6 h and also through the exposure of them on solid metal surfaces (stainless steel and copper coupons) for 75 min, wherein the copper sensitivity and resistance of the clinical isolates of Acinetobacter spp. were determined. Results: There was no interference with the growth of the isolates at the low concentrations, whereas the bacterial growth was affected by the high concentrations of CuSO4 at different levels. During the exposure on the solid metal coupons, no loss of viability of isolates was observed on stainless steel, however, the rapid death of isolates was discernible on copper surface, leading to a dramatic decrease in the number of colony-forming units (CFUs), eventually to the limit of detection (3 CFUs per coupon). Conclusion: This study substantiated that copper possesses antimicrobial properties which can be deployed in novel therapies for the prevention of the infections caused by Acinetobacter spp. and other emerging pathogens. Further studies on the sensitivity and resistance of Acinetobacter spp. to copper at the molecular genetics levels can open the door to better exploitation of this metal for the inhibition of the vigorous growth of drug-resistant Acinetobacter spp.

PHENOTYPIC DETECTION OF EFFLUX MECHANISM IN AMIKACIN-RESISTANT ACINETOBACTER ISOLATES FROM TWO DIFFERENT STATES IN SOUTH INDIA

Acinetobacter has already gained resistance to the majority of antibiotics available. Aminoglycosides are commonly used to treat invasive infections. Aminoglycoside resistance is associated with decreased drug absorption, aminoglycoside modification, and aminoglycoside efflux. The aim of this study was to detect the presence of an efflux mechanism in amikacin-resistant Acinetobacter isolated from hospital wards using Carbonyl Cyanide 3- Chlorophenylhydrazone (CCCP). One hundred isolates of Acinetobacter were isolated from tertiary care hospitals in two distinct South Indian states. Antibacterial susceptibility patterns were discovered between 2017 and 2019. Amikacin minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates was determined using Clinical and Laboratory Standards Institute (CLSI) standards. The efflux system activity was determined using CCCP. Among 100 Acinetobacter baumannii isolates, 49 isolates with amikacin resistance were found. The MIC’s of Acinetobacter ranged between 2 – 1024 μg/mL for the amikacin studied. After treatment with the efflux pump inhibitor, 38.77% of isolates became less resistant to amikacin, as determined by phenotypic detection of efflux pumps, showing a decrease in antibiotic MICs of at least four fold. The data demonstrated the importance of efflux pump activity conferring amikacin resistance on Acinetobacter clinical isolates.

A New Twist: The Combination of Sulbactam/Avibactam Enhances Sulbactam Activity against Carbapenem-Resistant Acinetobacter baumannii (CRAB) Isolates.

An increasing number of untreatable infections are recorded every year. Many studies have focused their efforts on developing new β-lactamase inhibitors to treat multi-drug resistant (MDR) isolates. In the present study, sulbactam/avibactam and sulbactam/relebactam combination were tested against 187 multi-drug resistant (MDR) Acinetobacter clinical isolates; both sulbactam/avibactam and sulbactam/relebactam restored sulbactam activity. A decrease ≥2 dilutions in sulbactam MICs was observed in 89% of the isolates when tested in combination with avibactam. Sulbactam/relebactam was able to restore sulbactam susceptibility in 40% of the isolates. In addition, the susceptibility testing using twenty-three A. baumannii AB5075 knockout strains revealed potential sulbactam and/or sulbactam/avibactam target genes. We observed that diazabicyclooctanes (DBOs) β-lactamase inhibitors combined with sulbactam restore sulbactam susceptibility against carbapenem-resistant Acinetobacter clinical isolates. However, relebactam was not as effective as avibactam when combined with sulbactam. Exploring novel combinations may offer new options to treat Acinetobacter spp. infections, especially for widespread oxacillinases and metallo-β-lactamases (MBLs) producers.

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

BackgroundThis study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.MethodsAntimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR).ResultsOverall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected.ConclusionsOur findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

1451. Genetic Analysis of Antibiotic Resistance Profiles of Acinetobacter baumannii Using Whole Genome Sequencing

Background Acinetobacter is known to quickly develop resistance to commonly used antibiotics. Previously we performed whole genome sequencing (WGS) and whole genome multilocus sequence typing (wgMLST) analysis in clinical Acinetobacter isolates to determine sequence types (ST) of these isolates and mapped their distribution. In this study, we sought to characterize the genetic antibiotic resistance patterns in these isolates.MethodsSixty-two clinical Acinetobacter isolates collected in two distinct large tertiary care hospitals in Detroit were analyzed. The samples were subjected to WGS using the NextSeq instrument (Illumina). The contigs were de novo assembled using SPAdes (v3.7.1) and wgMLST analysis was performed using BioNumerics software v7.6. The genomic sequence for each isolate was uploaded in ResFinder 3.2 and known antibiotic resistance genes were analyzed.ResultsThe most common resistance gene found is blaADC-25 conferring resistance to beta-lactams across all STs. Sulfonamide (sul1) and macrolide resistance (mphE/msrE) among STs were also common. ST2 (52%) was predominant for both hospitals (H1 and H2). ST2 in H2 exhibited the presence of the maximum number of resistance genes including resistance to aminoglycosides, macrolides (2), tetracyclines (tetB), beta-lactams, fluoroquinolones (aac(6’)-Ib-cr), sulphonamides (sul1, sul2). ST2 had a slightly different resistance profile of beta-lactams in H1 when compared to H2. ST406 and ST15 exhibited similar antibiotic profiles in both hospitals and a single isolate of ST20 from H2 is highly antibiotic resistant.Table 1. Antimicrobial Drug Resistance Profiles of all Acinetobacter Sequence Types (STs) in H1 and H2. Table 2. Antibiotic Resistance Gene Profiles of Sequence Type 2 (ST2) isolates in H1 and H2. ConclusionThis study provides us with a snapshot of antibiotic resistant genes among circulating Acinetobacter isolates prevalent in an area. All hospital isolates demonstrated resistance to beta-lactams. Multidrug resistant ST2 isolates from both hospitals demonstrate similar antibiotic resistance gene profiles suggesting a common circulating strain in the area. No colistin resistance genes were detected in any isolates. Because Acinetobacter infections are predominantly hospital acquired, it is important to continually monitor resistance profiles to determine the trends that may better serve both the patients and infection control practices.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support)

Biofilm formation and multidrug resistance in nosocomial isolates of Acinetobacter

Purpose: To study the biofilm formation and to examine the correlation between antibiotic resistance and biofilm formation among the clinical isolates of Acinetobacter. Materials and Methods: A total of 43 isolates of Acinetobacter collected from samples like peripheral venous catheter tips, urine from Foley’s catheter, central venous catheter tips and endotracheal tube aspirates were preserved and processed. The tube method was performed to qualitatively detect Biofilm production. Antimicrobial susceptibility was done as per CLSI guidelines. Place of Study and Study Period: Yenepoya medical college, Mangaluru and June 2015 to December 2015. Study Design: Prospective Results: Out of 43 isolates of Acinetobacter, 26(60%) showed biofilm production, among which 19 isolates (73%) were Multidrug resistant (MDR) and only 7 isolates (27%) were Non-MDR. Highest sensitivity was seen to colistin (100%) followed by imipenem (73.7%) and least sensitivity to ampicillin (0%) followed by ciprofloxacin (3.8%). Conclusion: Antibiotic resistance was found to be significantly higher among biofilm producing Acinetobacter isolates than nonbiofilm isolates. Routine and advanced studies of the biofilm production will help in making better usage of the invasive devices with less critical complications. Keywords: Biofilms, Acinetobacter, Multidrug resistance.

In Vitro Activity of the Ultra-Broad-Spectrum Beta-Lactamase Inhibitor QPX7728 in Combination with Meropenem against Clinical Isolates of Carbapenem-Resistant Acinetobacter baumannii.

QPX7728 is a recently discovered ultra-broad-spectrum beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in beta-lactamase-producing Gram-negative bacteria, including Acinetobacter spp. The potency of meropenem alone and in combination with QPX7728 (1 to 16 μg/ml) was tested against 275 clinical isolates of Acinetobacter baumannii (carbapenem-resistant A. baumannii [CRAB]) collected worldwide that were highly resistant to carbapenems (MIC50 and MIC90 for meropenem, 64 and >64 μg/ml). Addition of QPX7728 resulted in a marked concentration-dependent increase in meropenem potency, with the MIC90 of meropenem alone decreasing from >64 μg/ml to 8 and 4 μg/ml when tested with fixed concentrations of QPX7728 at 4 and 8 μg/ml, respectively. In order to identify the mechanisms that modulate the meropenem-QPX7728 MIC, the whole-genome sequences were determined for 135 isolates with a wide distribution of meropenem-QPX7728 MICs. This panel of strains included 116 strains producing OXA carbapenemases (71 OXA-23, 16 OXA-72, 16 OXA-24, 9 OXA-58, and 4 OXA-239), 5 strains producing NDM-1, one KPC-producing strain, and 13 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 μg/ml). Our analysis indicated that mutated PBP3 (with mutations localized in the vicinity of the substrate/inhibitor binding site) is the main factor that contributes to the reduction of meropenem-QPX7728 potency. Still, >90% of isolates that carried PBP3 mutations remained susceptible to ≤8 μg/ml of meropenem when tested with a fixed 4 to 8 μg/ml of QPX7728. In the absence of PBP3 mutations, the MICs of meropenem tested in combination with 4 to 8 μg/ml of QPX7728 did not exceed 8 μg/ml. In the presence of both PBP3 and efflux mutations, 84.6% of isolates were susceptible to ≤8 μg/ml of meropenem with 4 or 8 μg/ml of QPX7728. The combination of QPX7728 with meropenem against CRAB isolates with multiple resistance mechanisms has an attractive microbiological profile.

Metallo-Beta-Lactamase-Producing Acinetobacter spp. from Clinical Isolates at a Tertiary Care Hospital in Ghana.

Metallo-beta-lactamase-producing Acinetobacter spp. is a major challenge for therapeutic treatment of nosocomial infections. This study is aimed at determining the prevalence of MBL-producing Acinetobacter spp. among 87 clinical isolates of Acinetobacter spp. from the Korle-Bu Teaching Hospital, Accra, between August 2014 and July 2015. Acinetobacter spp. was identified by standard bacteriological method, and resistance to different antibiotics was assessed with the Kirby–Bauer disc diffusion method. Meropenem-resistant Acinetobacter isolates were screened for enzyme activity using the modified Hodge test (MHT) and combined disc test (CDT). Additionally, multiplex PCR was used to determine MBL genes presence (blaVIM,blaIMP, and blaNDM). All Acinetobacter isolates showed high resistance to cefotaxime (90.8%), ceftazidime (75.9%), cotrimoxazole (70.1%), ciprofloxacin (64.4%), gentamicin (72.4%), levofloxacin (67.8%), and meropenem (59.8%). A total of 54 (62.1%) of Acinetobacter isolates were multidrug-resistant. Out of 52 (59.8%) meropenem-resistant Acinetobacter, 3 (5.8%) were carbapenemase producers by MHT, whilst, 23 (44.2%) were CDT positive. There was no significant difference between the resistance pattern of amikacin, ceftazidime, cotrimoxazole, ciprofloxacin, and meropenem amongst CDT-positive and CDT-negative isolates (p > 0.05). A total of 7/87 (8.1%) CDT-positive Acinetobacter isolates harboured blaNDM; of these, 4 (57.1%) were from wound swabs, urine (n = 2) (28.6%), and ear swab (n = 1) (14.3%). The study revealed that less than 9% of Acinetobacter spp. contained blaNDM encoding genes. Strict antibiotics usage plan and infection control measures are required to prevent the spread of these resistance genes.

Role of AdeB gene in multidrug-resistance Acinetobacter

Objective The aim was to determine the role of the Ade B gene in multidrug-resistant Acinetobacter isolated from ICUs of Menoufia University Hospitals. Background Acinetobacter is a gram-negative bacteria that may cause serious infections. Numerous mechanisms are involved in its resistance to drug therapy. The active efflux mechanism is an important factor for development of multidrug resistance. The Ade ABC system is important efflux system in mediating such resistance. Therefore, the present study was designed to analyze the association between the expression level of Ade B gene and drug resistance in Acinetobacter. Materials and methods This case–control study was carried out in the period between October 2016 and October 2018. It was done at Clinical Pathology Department, Faculty of Medicine, Menoufia University Hospitals. The patients were selected from ICUs of Menoufia University Hospitals. The study included clinical samples collected from 614 patients admitted to ICUs. All clinical Acinetobacter isolates were further studied for determination of antibiotic susceptibility patterns and detection of Ade B gene by real-time PCR. Results Of the 614 samples, 70 (11.4%) Acinetobacter were isolated. Regarding antimicrobial resistance pattern, 61.4% of the Acinetobacter isolates were found multidrug and extensive drug resistant. There was significant increase in Ade B gene expression (P Conclusion AdeB gene plays a vital role in multidrug resistance in clinical Acinetobacter isolates. These results may benefit to design active efflux pump inhibitors. Moreover, implementation of strict microbial policies and infection control programs may prevent the rapid dissemination of this organism.

Determination of imipenem efflux-mediated resistance in Acinetobacter spp., using an efflux pump inhibitor

In recent years, reports of Acinetobacter strains resistant to all known antibiotics have caused a great concern in medical communities. Overexpression of efflux pumps is one of the major causes of resistance in bacteria. The aim of this study was to investigate the role of efflux pumps in conferring resistance to imipenem in clinically important Acinetobacter spp; Acinetobacter baumannii and Acinetobacter lwoffii. A total number of 46 clinical Acinetobacter isolates, including 33 A. baumannii and 13 A. lwoffii isolates, previously collected from Shahid Kamyab and Ghaem hospitals of Mashhad, Iran were used in this study. Imipenem susceptibility testing was carried out by the disc diffusion method. Imipenem minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates were determined both in the presence and absence of the efflux pumps inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Resistance to imipenem was observed in 38 isolates including 30 A. baumannii and 8 A. lwoffii isolates. Experiments in the presence of CCCP showed a 2 to 16384 fold reduction in imipenem MICs in 14 A. baumannii and 2 A. lwoffii isolates. The results obtained showed high levels of resistance to imipenem and contribution of efflux pumps in conferring resistance in both Acinetobacter species in this study. Moreover, imipenem efflux mediated resistance highlights the importance of this mechanism not only in A. baumannii but also in non-baumannii Acinetobacter Spp. which have been neglected in antibiotic resistance studies.

Clonal Dissemination of Clinical Isolates of Acinetobacter baumannii Carriers of 16S rRNA Methylase Genes in an Oncological Hospital in Recife, Brazil.

16S rRNA methylases confer high-level resistance to aminoglycosides which are used to treat serious infections caused by gram-negative bacteria, such as Acinetobacter spp. Some genes encoding these enzymes are disseminated worldwide, while others were detected in only some countries. The objective was to characterize the susceptibility profile to aminoglycosides (amikacin and gentamicin) of clinical isolates of Acinetobacter spp. from an oncological hospital in Recife, and given the resistance to both antimicrobials, to characterize minimal inhibitory concentrations (MICs) of amikacin, gentamicin and tobramycin, the occurrence of 16S rRNA methylase genes (armA, rmtB, rmtC and rmtD) and of ß-lactamase gene (blaKPC) and the clonal profile. Isolates resistant to both antimicrobials, amikacin and gentamicin, were selected by disk diffusion technique in Mueller-Hinton agar and identified. Broth microdilution was conducted to determine MICs of amikacin, gentamicin, and tobramycin. These isolates were subjected to polymerase chain reaction and pulsed-field gel electrophoresis. Among 23 analyzed isolates, 12 (52.2%) were resistant to gentamicin and amikacin and identified as Acinetobacter baumannii. Among these, 11 (91.7%), 12 (100%), and 9 (75%) isolates showed respectively MICs >256 µg/mL of amikacin, >64 µg/mL of gentamicin, and >64 µg/mL of tobramycin. The armA gene was found in 12 (100%) isolates and 6 (50%) showed coexistence of armA, rmtB, and rmtC genes. The rmtD and blaKPC genes were not detected. These isolates showed high genetic similarity (92%) and were classified as clone A. Elaboration and fulfillment of measures are thus essential to prevent the spread of this resistance mechanism.

Optimizing a Whole-Genome Sequencing Data Processing Pipeline for Precision Surveillance of Health Care-Associated Infections.

The surveillance of health care-associated infection (HAI) is an essential element of the infection control program. While whole-genome sequencing (WGS) has widely been adopted for genomic surveillance, its data processing remains to be improved. Here, we propose a three-level data processing pipeline for the precision genomic surveillance of microorganisms without prior knowledge: species identification, multi-locus sequence typing (MLST), and sub-MLST clustering. The former two are closely connected to what have widely been used in current clinical microbiology laboratories, whereas the latter one provides significantly improved resolution and accuracy in genomic surveillance. Comparing to a broadly used reference-dependent alignment/mapping method and an annotation-dependent pan-/core-genome analysis, we implemented our reference- and annotation-independent, k-mer-based, simplified workflow to a collection of Acinetobacter and Enterococcus clinical isolates for tests. By taking both single nucleotide variants and genomic structural changes into account, the optimized k-mer-based pipeline demonstrated a global view of bacterial population structure in a rapid manner and discriminated the relatedness between bacterial isolates in more detail and precision. The newly developed WGS data processing pipeline would facilitate WGS application to the precision genomic surveillance of HAI. In addition, the results from such a WGS-based analysis would be useful for the precision laboratory diagnosis of infectious microorganisms.

Dissemination of carbapenem-resistant Acinetobacter baumannii strains carrying the bla GES, bla NDM and bla OXA23 in Morocco.

Acinetobacter baumannii is a microorganism which has been classified by the World Health Organization in the list of the bacterial strains that pose the biggest danger to human health. This study was performed to determine the susceptibility profile to carbapenems and to detect carbapenemases production in 111 A. baumannii isolates. Among these 30 are environmental isolates and 81 are from the three major hospitals in Morocco. All strains of A. baumannii were tested against diverse antimicrobial agents (13 antibiotic drugs) by the agar diffusion test. Minimum inhibitory concentration (MIC) of imipenem on carbapenem-resistant strains (CRAB) was determined by the E-test technique. Simple phenotypic tests were used to detect carbapenemases and metallo-β-lactamases (MBLs) production including the modified Hodge test, EDTA test, and the cloxacillin test. The presence of carbapenemases-encoding resistance genes of CRAB strains was examined using polymerase chain reaction (PCR). Carbapenem resistance was observed in 23 clinical Acinetobacter isolates showing dissemination of the multiresistance profile. Molecular biology techniques indicated that all these strains encoded the naturally occurring bla OXA-51-like gene and were proved as A. baumannii. The bla OXA-23 gene was detected in 16 strains (69.6%). The metallo-β-lactamase bla NDM gene was detected in five isolates (21.7%). GES-type carbapenemases were found in 15 strains, the existence of three classes of carbapenemases (bla GES, bla NDM, and bla OXA-23) was detected in three strains, while none of the CRAB isolates contained the bla OXA-58, bla OXA-24, bla VIM, bla OXA-48 or bla KPC encoding genes. This study established baseline proof of three classes of carbapenemases producing A. baumannii in Morocco, showing the important role of surveillance in controlling their spread.

Molecular Epidemiology of Emerging Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter pittii in Taiwan, 2010 to 2014.

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