Carbapenem Resistance in Acinetobacter calcoaceticus-baumannii Complex Isolates From Kathmandu Model Hospital, Nepal, Is Attributed to the Presence of bla OXA-23-like and bla NDM-1 Genes.

The Acinetobacter calcoaceticus-baumannii (ACB) complex, also known as ACB complex, consists of four bacterial species that can cause opportunistic infections in humans, especially in hospital settings. Conventional therapies for susceptible strains of the ACB complex include broad-spectrum cephalosporins, ß-lactam/ß-lactamase inhibitors, and carbapenems. Unfortunately, the effectiveness of these antibiotics has declined due to increasing rates of resistance. The predominant resistance mechanisms identified in the ACB complex involve carbapenem-resistant (CR) oxacillinases and metallo-ß-lactamases (MBLs). This research, conducted at Kathmandu Model Hospital in Nepal, sought to identify genes associated with CR, specifically blaNDM-1, blaOXA-23-like, and blaOXA-24-like genes in carbapenem-resistant Acinetobacter calcoaceticus-baumannii (CR-ACB) complex. Additionally, the study is aimed at identifying the ACB complex through the sequencing of the 16s rRNA gene. Among the 992 samples collected from hospitalized patients, 43 (approximately 4.334%) tested positive for the ACB complex. These positive samples were mainly obtained from different hospital units, including intensive care units (ICUs); cabins; and neonatal, general, and maternity wards. The prevalence of infection was higher among males (58.14%) than females (41.86%), with the 40-50 age group showing the highest infection rate. In susceptibility testing, colistin and polymyxin B exhibited a susceptibility rate of 100%, whereas all samples showed resistance to third-generation cephalosporins. After polymyxins, gentamicin (30.23%) and amikacin (34.88%) demonstrated the highest susceptibility. A substantial majority (81.45%) of ACB complex isolates displayed resistance to carbapenems, with respiratory and pus specimens being the primary sources. Polymerase chain reaction (PCR) revealed that the primary CR gene within the ACB complex at this hospital was bla OXA-23-like, followed by bla NDM-1. To ensure the accuracy of the phenotypic assessment, 12 samples were chosen for 16s rRNA sequencing using Illumina MiSeq™ to confirm that they are Acinetobacter species. QIIME 2.0 analysis confirmed all 12 isolates to be Acinetobacter species. In the hospital setting, a substantial portion of the ACB complex carries CR genes, rendering carbapenem ineffective for treatment.

Colorimetric Loop-Mediated Isothermal Amplification Assays Accurately Detect blaOXA-23-like and ISAba1 Genes from Acinetobacter baumannii in Pure Cultures and Spiked Human Sera.

Carbapenem resistance in Acinetobacter baumannii is a critical global health threat attributed to transferrable carbapenemase genes. Carbapenemase genotyping using polymerase chain reaction (PCR) presents a challenge in resource-limited settings because of its technical requirements. This study designed new loop-mediated isothermal amplification (LAMP) primers using multiple sequence alignment-based workflows, validated the primer performance against multiple target variants in silico, and developed novel LAMP assays (LAntRN-OXA23 and LAntRN-ISAba1) to detect the transferable blaOXA-23-like carbapenemase genes and ISAba1 elements in pure cultures and A. baumannii-spiked serum samples. The designed LAMP primers bind to the conserved regions of their highly polymorphic targets, with their in silico performance comparable with other published primers. The in vitro LAMP assays (using 30 PCR-profiled A. baumannii and 10 standard multidrug-resistant gram-negative isolates) have 100% concordance with the PCR-positive clinical samples, limits of detection as low as 1 pg/µL (200 copies/µL), and specificities of 57.89-100%. Both assays produced positive results when testing DNA samples (extracted using a commercial kit) from blaOXA-23-like and ISAba1-blaOXA-51-like PCR-positive A. baumannii-spiked normal human sera (five set-ups per target). In summary, the LAMP assays accurately detected the target genes and have applications in infection management, control, and point-of-care testing in resource-limited healthcare settings.

Whole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure "ISAba1-blaADC-25." The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.IMPORTANCEThe blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

Multicenter retrospective genomic characterization of carbapenemase-producing Acinetobacter baumannii isolates from Jiangxi patients 2021-2022: identification of a novel international clone, IC11.

This study aimed to characterize carbapenem-resistant Acinetobacter baumannii (CRAB) isolates from Jiangxi patients using whole-genome sequencing (WGS). We subjected 100 clinical CRAB strains isolated from the three local largest teaching hospitals to WGS and antimicrobial susceptibility testing. Molecular epidemiology was investigated using multilocus sequence typing, core genome multilocus typing, core genome single-nucleotide polymorphism phylogeny, and pulsed-field gel electrophoresis. The most prevalent acquired carbapenemase was blaOXA-23, predominant in all isolates (100%). Isolates belonging to the dominating international clone IC2 accounted for 92% of all isolates. International IC11 (ST164Pas/ST1418Ox) clone was found in an additional 8% (eight isolates), with seven isolates (87.5%) carrying an acquired additional blaNDM-1 carbapenemase. The oxa23-associated Tn2009, either alone or in a tandem repeat structure containing four copies of blaOXA-23, was discovered in 62% (57 isolates) of IC2. The oxa23-associated Tn2006 was identified in 38% (35 isolates) of IC2 and all IC11 isolates. A putative conjugative RP-T1 (formerly RepAci6) plasmid with blaOXA-23 in Tn2006 within AbaR4, designated pSRM1.1, was found in IC2 A. baumannii strain SRM1. The blaNDM-1 gene found in seven IC11 isolates was located on a novel Tn6924-like transposon, a first-time report in IC11. These findings underscore the significant importance of real-time surveillance to prevent the further spread of CRAB. IMPORTANCE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is notorious for causing difficult-to-treat infections. To elucidate the molecular and clinical epidemiology of CRAB in Jiangxi, clinical CRAB isolates were collected and underwent whole-genome sequencing and antibiotic susceptibility phenotyping. Key findings included the predominance of OXA-23-producing IC2 A. baumannii, marked by the emergence of OXA-23 and NDM-1-producing IC11 strains.

Outbreak caused by pandrug-resistant blaOXA-69/blaOXA-23/blaGES harboring Acinetobacter baumannii ST2 in an intensive care unit.

Acinetobacter baumannii has emerged as a main nosocomial pathogen exhibiting high rates of resistance to clinically relevant antibiotics. Six pandrug-resistant A. baumannii (PDR-A. baumannii) were recovered from three patients in a Tunisian Intensive Care Unit (ICU) between 10th and 16th of May 2018 resulting in one fatal case and raising the possibility of an outbreak. On 18th of May environmental screening of ICU surfaces was carried out. On 22nd of May a fourth patient was infected with PDR-A. baumannii and died. A second investigation was carried out for environmental screening and PDR-A. baumannii was isolated from the respirator. Antimicrobial susceptibility testing was performed according to EUCAST (2019) guidelines. MIC of colistin was determined by broth microdilution method. PCR was used to detect 14 beta-lactamases/carbapenemases and mcr (mcr-1 to mcr-5) genes. The genetic relatedness of PDR-A. baumannii isolates was determined by PFGE and MLST. Seven PDR-A. baumannii isolates were recovered from four patients, one MDR strain from wash basin, a PDR strain from hand sanitizer bottle and another PDR strain from respirator. All PDR-A. baumannii (n = 9) harbored blaOXA-69 gene and none carried mcr. Moreover, seven carried blaGES and blaOXA-23 genes. PFGE identified four pulsotypes (A, B, C, and D) with the pulsotype A gathering seven PDR-A. baumannii isolates: six from three patients and one from hygiene sample. MLST revealed that all PDR-A. baumannii isolates of pulsotype A belonged to the pandemic clone ST2. Systematic screening of MDR and PDR-A. baumannii is highly recommended to limit dissemination of such strains in ICUs.

Molecular and clinical epidemiology of carbapenem resistant Acinetobacter baumannii ST2 in Oceania: a multicountry cohort study.

Background: Carbapenem resistant Acinetobacter baumannii (CRAb) is categorised by the World Health Organization (WHO) as a pathogen of critical concern. However, little is known about CRAb transmission within the Oceania region. This study addresses this knowledge gap by using molecular epidemiology to characterise the phylogenetic relationships of CRAb isolated in hospitals in Fiji, Samoa, and other countries within the Oceania region including Australia and New Zealand, and India from South Asia. Methods: In this multicountry cohort study, we analysed clinical isolates of CRAb collected from the Colonial War Memorial Hospital (CWMH) in Fiji from January through December 2019 (n = 64) and Tupua Tamasese Mea'ole Hospital (TTMH) in Samoa from November 2017 through June 2021 (n = 32). All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole-genome sequencing. For CWMH, data were collected on clinical and demographic characteristics of patients with CRAb, duration of hospital stay, mortality and assessing the appropriateness of meropenem use from the treated patients who had CRAb infections. To provide a broader geographical context, CRAb strains from Fiji and Samoa were compared with CRAb sequences from Australia collected in 2016-2018 (n = 22), New Zealand in 2018-2021 (n = 13), and India in 2019 (n = 58), a country which has close medical links with Fiji. Phylogenetic relationships of all these CRAb isolates were determined using differences in core genome SNPs. Findings: Of CRAb isolates, 49 (77%) of 64 from Fiji and all 32 (100%) from Samoa belonged to CRAb sequence type 2 (ST2). All ST2 isolates from both countries harboured blaOXA-23, blaOXA-66 and ampC-2 genes, mediating resistance to ß-lactam antimicrobials, including cephalosporins and carbapenems. The blaOXA-23 gene was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006, on the chromosome. Two distinct clusters (group 1 and group 2) of CRAb ST2 were detected in Fiji. The first group shared common ancestral linkage to all CRAb ST2 collected from Fiji's historic outbreak in 2016/2017, Samoa, Australia and 54% of total New Zealand isolates; they formed a single cluster with a median (range) SNP difference of 13 (0-102). The second group shared common ancestral linkage to 3% of the total CRAb ST2 isolated from India. Fifty eight of the 64 patients with CRAb infections at the CWMH had their first positive CRAb sample collected 72 h or more following admission. Meropenem use was deemed inappropriate in 15 (48%) of the 31 patients that received treatment with meropenem in Fiji. Other strains of CRAb ST1, ST25, ST107, and ST1112 were also detected in Fiji. Interpretation: We identified unrecognised outbreaks of CRAb ST2 in Fiji and Samoa that linked to strains in other parts of Oceania and South Asia. The existence of Tn2006, containing the blaOXA-23 and ISAba1 insertion element, within CRAb ST2 from Fiji and Samoa indicates the potential for high mobility and dissemination. This raises concerns about unmitigated prolonged outbreaks of CRAb ST2 in the two major hospitals in Fiji and Samoa. Given the magnitude of this problem, there is a need to re-evaluate the current strategies used for infection prevention and control, antimicrobial stewardship, and public health measures locally and internationally. Moreover, a collaborative approach to AMR surveillance within the Oceania region with technical, management and budgetary support systems is required to prevent introduction and control transmission of these highly problematic strains within the island nation health systems. Funding: This project was funded by an Otago Global Health Institute seed grant and Maurice Wilkins Centre of Research Excellence (CoREs) grant (SC0000169653, RO0000002300).

Healthcare Equipment and Personnel Reservoirs of Carbapenem-Resistant Acinetobacter baumannii Epidemic Clones in Intensive Care Units in a Tunisian Hospital.

Carbapenem-resistant Acinetobacter baumannii (CRAB) strains can cause severe and difficult-to-treat infections in patients with compromised general health. CRAB strains disseminate rapidly in nosocomial settings by patient-to-patient contact, through medical devices and inanimate reservoirs. The occurrence of CRAB in patients residing in the intensive care units (ICUs) of the Sahloul University hospital in Sousse, Tunisia is high. The objective of the current study was to determine whether the surfaces of items present in five ICU wards and the medical personnel there operating could serve as reservoirs for CRAB strains. Furthermore, CRAB isolates from patients residing in the ICUs during the sampling campaign were analyzed for genome comparison with isolates from the ICUs environment. Overall, 206 items were screened for CRAB presence and 27 (14%) were contaminated with a CRAB isolate. The items were located in several areas of three ICUs. Eight of the 54 (15%) screened people working in the wards were colonized by CRAB on the hands. Patients residing in the ICUs were infected with CRAB strains sharing extensive genomic similarity with strains recovered in the nosocomial environment. The strains belonged to three sub-clades of the internationally disseminated clone (ST2). A clone emerging in the Mediterranean basin (ST85) was detected as well. The strains were OXA-23 or NDM-1 producers and were also pan-aminoglycoside resistant due to the presence of the armA gene. Hygiene measures are urgent to be implemented in the Sahloul hospital to avoid further spread of difficult-to-treat CRAB strains and preserve health of patients and personnel operating in the ICU wards.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in Turkiye: Systematic review.

The World Health Organization has designated carbapenem-resistant Acinetobacter baumannii (CRAB) as a "critical" pathogen on the global priority list of antibiotic-resistant bacteria. This study aims to discuss the molecular epidemiology of CRAB isolates in Turkiye in the last 12 years and the prevalence of gene regions associated with resistance or pathogenesis using a systematic review method. Our study consists of a literature search, determination of eligibility and exclusion criteria, qualitative analysis of studies, data extraction, and statistical analysis. All studies were analyzed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Guidelines. The incidence rates of blaOXA-23, blaOXA-23-like, blaOXA-24/40, blaOXA-24/40-like, blaOXA-51, blaOXA-51-like, blaOXA-58, and blaOXA-58-like genes in CRAB strains were 76.4%, 68.6%, 1.2%, 3.4%, 97.0%, 98.6%, 8.4%, and 17.1%, respectively. It was determined that the prevalence of the blaOXA-23 and blaOXA-58 gene regions showed a statistically significant change over the years. Due to the high prevalence of A. baumannii strains carrying the blaOXA-23 variant, it is necessary to follow its geographical distribution and transposon and plasmid movements. Based on available data, molecular surveillance of CRAB strains should be standardized. In addition, sterilization and disinfection processes applied within the scope of an effective struggle against CRAB strains that can remain live on surfaces for a long time should be reviewed frequently.

Detection of blaoxa-23 Gene from Carbapenem-resistant Acinetobacter Baumannii.

BACKGROUND: Antibiotic resistance is a great concern for public health and Acinetobacter baumannii-associated infections are increasing in many parts of the world, including Nepal. However, limited data is available on the prevalence of A. baumannii harboring blaOXA-23 from Nepal. METHODS: A hospital-based cross-sectional study was designed to detect the blaOXA-23 gene from carbapenem-resistant A. baumannii isolates in Nepal. A total of 380 clinical specimens were collected and processed following standard microbiological procedures. Antibiotic susceptibility test was performed as per the protocol of the Kirby-Bauer disk diffusion technique and the CLSI guidelines, while screening of carbapenemase production was assessed by the Modified Hodge Test using meropenem (10µg) disc. The presence of the blaOXA-23 gene in carbapenemase-positive A. baumannii was confirmed by PCR. RESULTS: Among 380 specimens analyzed, 210 (55.3%) samples were positive for bacterial growth, where 33(15.7% of total growth) of the isolates were A. baumannii, and most of them were isolated from the ICU patients (20/33, 60.6%) and sputum (16/33, 48.5%). Thirty-two isolates (97%) were colistin sensitive, while only four (12.1%) isolates were sensitive to meropenem and imipenem. Twenty-three (69.7%) of A. baumannii were carbapenemase positive as revealed by the Modified Hodge Test test, and 19 of them (57.6% of total A. baumannii) harbored the blaOXA-23 gene. CONCLUSIONS: A high prevalence of the blaOXA-23 gene among carbapenem-resistant A. baumannii isolates were found. Systematic network surveillance should be established to check the spread of such isolates, especially in the intensive care units of tertiary care hospitals in Nepal.

High Genetic Diversity of Carbapenem-Resistant Acinetobacter baumannii Isolates Recovered in Nigerian Hospitals in 2016 to 2020.

Acinetobacter baumannii causes difficult-to-treat infections mostly among immunocompromised patients. Clinically relevant A. baumannii lineages and their carbapenem resistance mechanisms are sparsely described in Nigeria. This study aimed to characterize the diversity and genetic mechanisms of carbapenem resistance among A. baumannii strains isolated from hospitals in southwestern Nigeria. We sequenced the genomes of all A. baumannii isolates submitted to Nigeria's antimicrobial resistance surveillance reference laboratory between 2016 and 2020 on an Illumina platform and performed in silico genomic characterization. Selected strains were sequenced using the Oxford Nanopore technology to characterize the genetic context of carbapenem resistance genes. The 86 A. baumannii isolates were phylogenetically diverse and belonged to 35 distinct Oxford sequence types (oxfSTs), 16 of which were novel, and 28 Institut Pasteur STs (pasSTs). Thirty-eight (44.2%) isolates belonged to none of the known international clones (ICs). Over 50% of the isolates were phenotypically resistant to 10 of 12 tested antimicrobials. The majority (n = 54) of the isolates were carbapenem resistant, particularly the IC7 (pasST25; 100%) and IC9 (pasST85; >91.7%) strains. blaOXA-23 (34.9%) and blaNDM-1 (27.9%) were the most common carbapenem resistance genes detected. All blaOXA-23 genes were carried on Tn2006 or Tn2006-like transposons. Our findings suggest that a 10-kb Tn125 composite transposon is the primary means of blaNDM-1 dissemination. Our findings highlight an increase in blaNDM-1 prevalence and the widespread transposon-facilitated dissemination of carbapenemase genes in diverse A. baumannii lineages in southwestern Nigeria. We make the case for improving surveillance of these pathogens in Nigeria and other understudied settings. IMPORTANCE Acinetobacter baumannii bacteria are increasingly clinically relevant due to their propensity to harbor genes conferring resistance to multiple antimicrobials, as well as their ability to persist and disseminate in hospital environments and cause difficult-to-treat nosocomial infections. Little is known about the molecular epidemiology and antimicrobial resistance profiles of these organisms in Nigeria, largely due to limited capacity for their isolation, identification, and antimicrobial susceptibility testing. Our study characterized the diversity and antimicrobial resistance profiles of clinical A. baumannii in southwestern Nigeria using whole-genome sequencing. We also identified the key genetic elements facilitating the dissemination of carbapenem resistance genes within this species. This study provides key insights into the clinical burden and population dynamics of A. baumannii in hospitals in Nigeria and highlights the importance of routine whole-genome sequencing-based surveillance of this and other previously understudied pathogens in Nigeria and other similar settings.

Healthcare-Associated Bloodstream Infections Due to Multidrug-Resistant Acinetobacter baumannii in COVID-19 Intensive Care Unit: A Single-Center Retrospective Study.

Healthcare-associated infections are an emerging cause of morbidity and mortality in COVID-19 intensive care units (ICUs) worldwide, especially those caused by multidrug-resistant (MDR) pathogens. The objectives of this study were to assess the incidence of bloodstream infections (BSIs) among critically ill COVID-19 patients and to analyze the characteristics of healthcare-associated BSIs due to MDR Acinetobacter baumannii in an COVID-19 ICU. A single-center retrospective study was conducted at a tertiary hospital during a 5-month period. The detection of carbapenemase genes was performed by PCR and genetic relatedness by pulsed-field gel electrophoresis (PFGE) and multilocus-sequence typing. A total of 193 episodes were registered in 176 COVID-19 ICU patients, with an incidence of 25/1000 patient-days at risk. A. baumannii was the most common etiological agent (40.3%), with a resistance to carbapenems of 100%. The blaOXA-23 gene was detected in ST2 isolates while the blaOXA-24 was ST636-specific. PFGE revealed a homogeneous genetic background of the isolates. The clonal spread of OXA-23-positive A. baumannii is responsible for the high prevalence of MDR A. baumannii BSIs in our COVID-19 ICU. Further surveillance of resistance trends and mechanisms is needed along with changes in behavior to improve the implementation of infection control and the rational use of antibiotics.

Genomic Comparative Analysis of Two Multi-Drug Resistance (MDR) Acinetobacter baumannii Clinical Strains Assigned to International Clonal Lineage II Recovered Pre- and Post-COVID-19 Pandemic.

BACKGROUND: After the emergence of COVID-19, numerous cases of A. baumannii/SARS-CoV-2 co-infection were reported. Whether the co-infecting A. baumannii strains have distinctive characteristics remains unknown. METHODS AND RESULTS: A. baumannii AMA_NO was isolated in 2021 from a patient with COVID-19. AMA166 was isolated from a mini-BAL used on a patient with pneumonia in 2016. Both genomes were similar, but they possessed 337 (AMA_NO) and 93 (AMA166) unique genes that were associated with biofilm formation, flagellar assembly, antibiotic resistance, secretion systems, and other functions. The antibiotic resistance genes were found within mobile genetic elements. While both strains harbored the carbapenemase-coding gene blaOXA-23, only the strain AMA_NO carried blaNDM-1. Representative functions coded for by virulence genes are the synthesis of the outer core of lipooligosaccharide (OCL5), biosynthesis and export of the capsular polysaccharide (KL2 cluster), high-efficiency iron uptake systems (acinetobactin and baumannoferrin), adherence, and quorum sensing. A comparative phylogenetic analysis including 239 additional sequence type (ST) 2 representative genomes showed high similarity to A. baumannii ABBL141. Since the degree of similarity that was observed between A. baumannii AMA_NO and AMA166 is higher than that found among other ST2 strains, we propose that they derive from a unique background based on core-genome phylogeny and comparative genome analysis. CONCLUSIONS: Acquisition or shedding of specific genes could increase the ability of A. baumannii to infect patients with COVID-19.

Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the blaOXA-51 and blaOXA-23 genes of A. baumannii. The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 100-101 CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A. baumannii and CRAB.

Novel 16S rRNA methyltransferase RmtE3 in Acinetobacter baumannii ST79.

Introduction. The 16S rRNA methyltransferase (16S RMTase) gene armA is the most common mechanism conferring high-level aminoglycoside resistance in Acinetobacter baumannii, although rmtA, rmtB, rmtC, rmtD and rmtE have also been reported.Hypothesis/Gap statement. The occurrence of 16S RMTase genes in A. baumannii in the UK and Republic of Ireland is currently unknown.Aim. To identify the occurrence of 16S RMTase genes in A. baumannii isolates from the UK and the Republic of Ireland between 2004 and 2015.Methodology. Five hundred and fifty pan-aminoglycoside-resistant A. baumannii isolates isolated from the UK and the Republic of Ireland between 2004 and 2015 were screened by PCR to detect known 16S RMTase genes, and then whole-genome sequencing was conducted to screen for novel 16S RMTase genes.Results. A total of 96.5 % (531/550) of isolates were positive for 16S RMTase genes, with all but 1 harbouring armA (99.8 %, 530/531). The remaining isolates harboured rmtE3, a new rmtE variant. Most (89.2 %, 473/530) armA-positive isolates belonged to international clone II (ST2), and the rmtE3-positive isolate belonged to ST79. rmtE3 shared a similar genetic environment to rmtE2 but lacked an ISCR20 element found upstream of rmtE2.Conclusion. This is the first report of rmtE in A. baumannii in Europe; the potential for transmission of rmtE3 to other bacterial species requires further research.

Co-Occurrence of blaOXA-23 in the Chromosome and Plasmid: Increased Fitness in Carbapenem-Resistant Acinetobacter baumannii.

This study aims to explore the co-occurrence of chromosomal and plasmid blaOXA-23 in carbapenem-resistant A. baumannii (CRAB) and its influence on phenotypes. A total of 11 CRAB isolates containing copies of blaOXA-23 on the chromosome and plasmid (CO), as well as 18 closely related isolates with blaOXA-23, located on either the chromosome or plasmid (SI), were selected for the determination of antibiotic susceptibility, virulence phenotype, and characteristic genomic differences. The co-occurrence of blaOXA-23 on the CRAB chromosome and plasmids did not enhance carbapenem resistance, but trimethoprim/sulfamethoxazole exhibited significantly reduced minimum inhibitory concentrations in CO. CO demonstrated a higher degree of fitness compared to SI. An increased biofilm formation ability and serum tolerance were also identified in CO, which may be associated with virulence genes, which include csuD, entE, pgaA, and plc. blaOXA-23-carrying transposons were found at different insertion sites on the chromosome. The most common site was AbaR-type genomic islands (50%). Two types of plasmids were found in CO. The co-occurrence of blaOXA-23 on the chromosome and a plasmid in CRAB had little effect on carbapenem susceptibility but was accompanied by increased fitness and virulence. Different origins and independent insertions of blaOXA-23-carrying transposons were identified in both the chromosomal and plasmid sequences.

The Role of Uniform Meropenem Usage in Acinetobacter baumannii Clone Replacement.

The dominant carbapenem resistant Acinetobacter baumannii harboring blaOXA-23-like carbapenemase was replaced by blaOXA-40-like carriers in a Hungarian tertiary-care center with high meropenem but relatively low imipenem use. We hypothesized that alterations in antibiotic consumption may have contributed to this switch. Our workgroup previous study examined the relation between resistance spiral and the antibiotic consumption, and the results suggest that the antibiotic usage provoked the increasing resistance in case of A. baumannii. We aimed at measuring the activity of imipenem and meropenem to compare the selection pressure exerted by the different carbapenems in time-kill assays. Strain replacement was confirmed by whole genome sequencing, core-genome multilocus sequence typing (cgMLST), and resistome analysis. Based on results of the time-kill assays, we found a significant difference between two different sequence-types (STs) in case of meropenem, but not in case of imipenem susceptibility. The newly emerged ST636 and ST492 had increased resistance level against meropenem compared to the previously dominant ST2 and ST49. On the other hand, the imipenem and colistin resistance profiles were similar. These results suggest, that the uniform meropenem usage may have contributed to A. baumannii strain replacement in our setting.

The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

BACKGROUND: Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. METHODS: A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. RESULTS: MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. CONCLUSIONS: Our investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. METHODS: In this study, a 15-µL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 µL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 µL-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. RESULTS: The detection limit of the bla OXA-51 gene by 15 µL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 µL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 µL RPA data for detecting bla OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. CONCLUSION: We demonstrated that the new 15 µL RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

The first nationwide multicenter study of Acinetobacter baumannii recovered in Serbia: emergence of OXA-72, OXA-23 and NDM-1-producing isolates.

BACKGROUND: The worldwide emergence and clonal spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern. The aim of this nationwide study was to investigate the prevalence of CRAB isolates in Serbia and to characterize underlying resistance mechanisms and their genetic relatedness. METHODS: Non-redundant clinical samples obtained from hospitalized patients throughout Serbia were included in the prospective, observational, multicenter study conducted from January to June 2018. Samples were initially screened for the presence of Acinetobacter baumannii-calcoaceticus (Acb) complex using conventional bacteriological techniques. Acb complexes recovered from clinical samples obtained from inpatients with confirmed bacterial infections were further evaluated for the presence of A. baumannii. Identification to the species level was done by the detection of the blaOXA-51 gene and rpoB gene sequence analysis. Susceptibility testing was done by disk diffusion and broth microdilution method. CRAB isolates were tested for the presence of acquired carbapenemases (blaOXA-24-like, blaOXA-23-like,blaOXA-58-like, blaOXA-143-like, blaIMP, blaVIM, blaGIM, blaSPM, blaSIM, blaNDM) by PCR. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: Acb complex was isolated in 280 out of 2401 clinical samples (11.6%). Overall, A. baumannii was identified in 237 out of 280 Acb complex (84.6%). CRAB prevalence was found to be 93.7% (237/222). The MIC50/MIC90 for imipenem and meropenem were 8/> 32 µg/mL and 16/> 32 µg/mL, respectively. Although susceptibility was high for colistin (95.7%; n = 227) and tigecycline (75.1%; n = 178), ten isolates (4.3%) were classified as pandrug-resistant. The following carbapenemases-encoding genes were found: 98 (44.2%) blaOXA-24-like, 76 (34.5%) blaOXA-23-like, and 7 (3.2%) blaNDM-1. PFGE analysis revealed six different clusters. MLST analysis identified three STs: ST2 (n = 13), ST492 (n = 14), and ST636 (n = 10). Obtained results evaluated that circulating CRAB clones in Serbia were as follows: blaOXA66/blaOXA23/ST2 (32.4%), blaOXA66/blaOXA23/blaOXA72/ST2 (2.7%), blaOXA66/blaOXA72/ST492 (37.8%), and blaOXA66/blaOXA72/ST636 (27.1%). CONCLUSION: This study revealed extremely high proportions of carbapenem resistance among A. baumannii clinical isolates due to the emergence of blaOXA-72, blaOXA-23, and blaNDM-1 genes among CRAB isolates in Serbia and their clonal propagation.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of Carbapenem-Resistant Acinetobacter baumannii Strains Harboring blaOXA-23, and the Epidemiological Survey of Clinical Isolates.

Acinetobacter baumannii is an important nosocomial pathogen in hospital-acquired infections, and carbapenem resistance has been increasingly observed worldwide. Oxacillinase production by blaOXA-23 is a predominant and prevalent carbapenem resistance mechanism of A. baumannii, especially in China. Rapid and specific detection of blaOXA-23 may offer valuable insight for administration of directed antimicrobial therapy. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP)-based method for identifying carbapenem-resistant A. baumannii (CRAB) harboring the blaOXA-23 gene. High-specificity primers for screening blaOXA-23 were designed and synthesized, and the LAMP reactions were performed. Clinical A. baumannii strains isolated from the Former 307th Hospital of People's Liberation Army were used to determine the sensitivity and specificity of this method compared with those of phenotypic antimicrobial susceptibility testing and the traditional PCR method. Multilocus sequence typing (MLST) was performed to investigate the epidemiology of the A. baumannii bacterial population. Compared with antimicrobial susceptibility testing, the sensitivity and specificity of LAMP in detecting blaOXA-23 were 88.4% and 97.7%, respectively. However, the LAMP method is much simpler and less time-consuming (within 60 minutes) than conventional PCR and phenotypic susceptibility testing. The 113 isolates could be clustered into 30 sequence types, and most strains (83/113) belonged to clonal complex (CC) 92, which is also the dominant CC in China. The LAMP-based method detected blaOXA-23 in a simpler manner and could provide rapid results for identifying CRAB. Consequently, blaOXA-23 may serve as a surrogate marker for the presence of CRAB in patients with serious infections in clinical practice.