Insertion Sequence ISAba1 Research Articles

Virulence potential of multidrug-resistant Acinetobacter baumannii isolates from COVID-19 patients on mechanical ventilation: The first report from Serbia.

Since the WHO declared the COVID-19 pandemic in March 2020, the disease has spread rapidly leading to overload of the health system and many of the patients infected with SARS-CoV-2 needed to be admitted to the intensive care unit (ICU). Around 10% of patients with the severe manifestation of COVID-19 need noninvasive or invasive mechanical ventilation, which represent a risk factor for Acinetobacter baumannii superinfection. The 64 A. baumannii isolates were recovered from COVID-19 patients admitted to ICU at General Hospital "Dr Laza K. Lazarević" Šabac, Serbia, during the period from December 2020 to February 2021. All patients required mechanical ventilation and mortality rate was 100%. The goal of this study was to evaluate antibiotic resistance profiles and virulence potential of A. baumannii isolates recovered from patients with severe form of COVID-19 who had a need for mechanical ventilation. All tested A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and levofloxacin according to the broth microdilution method and MDR phenotype was confirmed. In all tested isolates, representatives of international clone 2 (IC2) classified by multiplex PCR for clonal lineage identification, bla AmpC, bla OXA-51, and bla OXA-23 genes were present, as well as ISAba1 insertion sequence upstream of bla OXA-23. Clonal distribution of one dominant strain was found, but individual strains showed phenotypic differences in the level of antibiotic resistance, biofilm formation, and binding to mucin and motility. According to PFGE, four isolates were sequenced and antibiotic resistance genes as well as virulence factors genes were analyzed in these genomes. The results of this study represent the first report on virulence potential of MDR A. baumannii from hospital in Serbia.

Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production.

Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii. A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR. A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-β-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1. The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.

Molecular Characterisation of Carbapenemase-Producing Acinetobacter baumannii isolates from Hospitalised Patients in Iraq

Carbapenem-resistant Acinetobacter baumannii has been considered one of the major threats to patients worldwide. To evaluate carbapenemase in several clinical isolates using phenotypic and genotypic approaches. A total of 49 A. baumannii isolates were tested against imipenem and meropenem discs on Muller Hinton agar, then screened phenotypically through the modified Hodge test (MHT), combined disc test (CDT) and modified carbapenem inactivation method (mCIM). The tested isolates have been subjected to polymerase chain reaction (PCR) detection to identify some carbapenemase-encoding genes and one insertion sequence. The carbapenem resistance profile showed 96% and 94% resistance to imipenem and meropenem, respectively. MHT and mCIM were able to produce carbapenemase in 94% and 98% of isolates, respectively, while CDT was able to produce metallo-B-lactamase (MBL) only in 59.2% of isolates. The PCR amplification of blaOXA-51 has been observed in all isolates. We found blaOXA-23 in 98% of isolates. Insertion sequence ISAba1 was present in all positive blaOXA-23 strains (98%). A blaVIM gene encoding MBL was present in 71% of isolates, but none of the isolates has been positive for blaKPC and blaNDM. The high rate of carbapenem resistance in A. baumannii became a serious threat worldwide. Concerning phenotypic tests, mCIM was the most sensitive compared to MHT and CDT. This study established that blaOXA-23 and blaOXA-51 have been the most prevalent among class D carbapenemase, and blaVIM among class B carbapenemase. The present study suggests that there might be silent carbapenemase genes in carbapenem-sensitive strains.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Isolated from Intensive Care Unit Patients in Jordanian Hospitals.

Acinetobacter baumannii is a common cause of healthcare-associated infections (HAI) worldwide, mostly occurring in intensive care units (ICUs). Extended-spectrum beta lactamases (ESBL)-positive A. baumannii strains have emerged as highly resistant to most currently used antimicrobial agents, including carbapenems. The most common mechanism for carbapenem resistance in this species is β-lactamase-mediated resistance. Carbapenem-hydrolyzing class D oxacillinases are widespread among multidrug-resistant (MDR) A. baumannii strains. The present study was conducted to determine the presence and distribution of blaOXA genes among multidrug-resistant A. baumannii isolated from ICU patients and genes encoding insertion sequence (IS-1) in these isolates. Additionally, the plasmid DNA profiles of these isolates were determined. A total of 120 clinical isolates of A. baumannii from various ICU clinical specimens of four main Jordanian hospitals were collected. Bacterial isolate identification was confirmed by biochemical testing and antibiotic sensitivity was then assessed. PCR amplification and automated sequencing were carried out to detect the presence of blaOXA-51, blaOXA-23, blaOXA-24, and blaOXA-58 genes, and ISAba1 insertion sequence. Out of the 120 A. baumannii isolates, 95% of the isolates were resistant to three or more classes of the antibiotics tested and were identified as MDR. The most frequent resistance of the isolates was against piperacillin (96.7%), cephalosporins (97.5%), and β-lactam/β-lactamase inhibitor combinations antibiotics (95.8%). There were 24 (20%) ESBL-producing isolates. A co-existence of blaOXA-51 gene and ISAba1 in all the 24 ESBL-producing isolates was determined. In addition, in the 24 ESBL-producing isolates, 21 (87.5%) carried blaOXA-51 and blaOXA-23 genes, 1 (4.2%) carried blaOXA-51 and blaOXA-24, but all were negative for the blaOXA-58 gene. Plasmid DNA profile A and profile B were the most common (29%) in ESBL-positive MDR A. baumannii isolates while plasmid DNA profile A was the most common in the ESBL-negative isolates. In conclusion, there was an increase in prevalence of MDR-A. baumannii in ICU wards in Jordanian hospitals, especially those having an ESBL phenotype. Thus, identification of ESBL genes is necessary for the surveillance of their transmission in hospitals.

Dissemination and Genetic Relatedness of Multidrug-Resistant and Extensively Drug-Resistant Acinetobacter baumannii Isolates from a Burn Hospital in Iraq.

Acinetobacter baumannii is an aggressive opportunistic bacterial pathogen that causes severe nosocomial infections, especially among burn patients. An increasing number of hospitals-acquired infections have been reported all over the world. However, little attention has been paid to the relatedness between A. baumannii isolates from different hospital environments and patients. In this study, 27 isolates were collected from the Burn and Plastic Surgery Hospital of Al Sulaymaniyah City, Iraq, from January through December 2019 (11 from patients and 16 from the wards environment), identified to species level as A. baumannii using Vitek 2 system and molecular detection of 16S rRNA gene, and then confirmed by targeting the blaOXA-51 gene. Moreover, the isolates were characterized by means of automated antimicrobial susceptibility assay, antimicrobial-resistant patterns, a phenotypic method using a combined disk test, and molecular methods for the detection of class A and C β-lactamase genes, and finally, the genetic relatedness was classified. Antimicrobial susceptibility testing showed that 63% (17/27) of the retrieved A. baumannii isolates were extensively drug-resistant to 8/9 antimicrobial classes. Furthermore, 37% (10/27) of the isolates were classified as multidrug-resistant; 8 isolates exhibited similar resistant patterns and the other two isolates showed 2 different patterns, while resistance was greater in isolates from patients than from the ward environment. Combined disk test showed that two isolates contained extended-spectrum β-lactamase. All isolates carried blaTEM-1, and two copies of the blaCTX-1 gene were indicated in one isolate, while blaSHV was absent in all isolates. Twenty-four isolates carried the blaAmpC gene; among them, 3 isolates harbored the insertion sequence ISAba-1 upstream to the gene. Using Enterobacterial Repetitive Intergenic Consensus PCR, the isolates were clustered into 6 distinct types; among them, two clusters, each of four strains, were classified to contain isolates from both patients and environments. The clusters of similar genotypes were found in inpatients as well as the environments of different wards during time periods, suggesting transmission within the hospital. Identification of possible infection sources and controlling the transmission of these aggressive resistance strains should be strictly conducted.

In vitro activity of sulbactam-durlobactam against carbapenem-resistant Acinetobacter baumannii and mechanisms of resistance

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

Identification of Promoter Region Markers Associated With Altered Expression of Resistance-Nodulation-Division Antibiotic Efflux Pumps in Acinetobacter baumannii.

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with “experimentally verified,” “presumed,” and “probably irrelevant” degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the “experimentally verified” level was permitted for 42 isolates to 7–8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.

Characterization of carbapenem resistance in environmental samples and Acinetobacter spp. isolates from wastewater and river water in Poland

The aim of this study was to analyze the prevalence of carbapenem resistance genes in Acinetobacter spp. isolated from wastewater in a municipal WWTP and to determine their spread from treated wastewater to river water with the use of conventional and molecular microbiology methods (qualitative and quantitative PCR and metagenomic analysis). Samples of untreated and treated wastewater and samples of river water obtained upstream and downstream from the wastewater discharge point were collected in 3 seasons (February, June, and September) of 2019. Acinetobacter spp. isolates were obtained by the culture method on the CHROMagar™ Acinetobacter medium. Additionally, environmental DNA was extracted from the samples for metagenomic and qPCR analyses. The presence of beta-lactam resistance genes (Ambler class B and D), insertion sequence ISAba1, and class I, II, and III integron-integrase genes was determined, and the bacterial taxonomic structure and wastewater and river samples was analyzed. Out of the 301 isolates obtained on the CHROMagar™ Acinetobacter medium, 258 belonged to the genus Acinetobacter, including 21 isolates that were identified as Acinetobacter baumannii. The highest number of Acinetobacter spp. and A. baumannii isolates were obtained from wastewater and river water samples collected in June and September. The ISAba1/blaOXA-51 complex was identified in 13 isolates, which confirms the occurrence of carbapenem-resistance isolates in the analyzed samples. The number of Acinetobacter isolates carrying antibiotic resistance genes (ARGs) increased in river water samples collected downstream from the wastewater discharge point (48 out of 258 isolates – 18.6%) compared to river water samples collected upstream from the wastewater discharge point (34 out of 258 isolates – 13.2%), which suggests that WWTP is a source of pollution in the natural environment. The conducted research provides evidence that bacteria of the genus Acinetobacter may spread alarming beta-lactam resistance in the environment and, therefore, pose a serious epidemiological threat.

Genomic insights of Acinetobacter baumannii ST374 reveal wide and increasing resistome and virulome

WGS-based surveillance has significantly improved the ability to track global spread and emergence of multidrug-resistant clones of clinically relevant pathogens. In this study, we performed the genomic characterization and comparative analysis of an Acinetobacter baumannii (strain Ac56) belonging to the sequence type ST374, which was isolated for the first time in Brazil, in 1996. Genomic analysis of Ac56 predicted a total of 5373 genes, with 3012 being identical across nine genomes of A. baumannii isolates of ST374 from European, Asian, North and South American countries. GoeBURST analysis grouped ST374 lineages into clonal complex CC3 (international clone IC-III). Resistome analysis of ST374 clone predicted genes associated with resistance to heavy metals and clinically relevant beta-lactams and aminoglycosides antibiotics. In this regard, in two closely related A. baumannii strains, the intrinsic blaADC gene was linked to the insertion sequence ISAba1; including the Ac56 strain, where it has been possibly associated with intermediate susceptibility to meropenem. Other four carbapenem-resistant A. baumannii strains carried the ISAba1/blaOXA-23 gene array, which was associated with the transposon Tn2008 or with Tn2006 in an AbaR4-type resistance island. While most virulence genes were shared for A. baumannii strains of ST374, three isolates from Thailand harbored KL49 capsular loci, previously identified in the hypervirulent A. baumannii LAC-4 strain. Analysis of thirty-four predicted plasmids showed eight major groups, of which GR-6 (LN−1) and GR-2 (LN−2) were prevalent. All strains, including the earliest isolate Ac56 harbored at least one complete prophage, whereas none CRISPR-associated (cas) gene was detected. In summary, genomic data of A. baumannii ST374 reveal a potential of this lineage to become a successful clone.

Evaluation of the occurrence of genetic determinants of multi-drug resistance among Acinetobacter baumannii strains

Introduction: The aim of the study was the analysis of occurrence of genetic determinants of multi-drug resistance and the assessment of genetic relationship among Acinetobacter baumannii strains. Methods: Multiplex-PCR method was performed in order to: (1) confirm the phenotypic identification and (2) detect the presence of CHDL oxacillinases in the group of thirty A.baumannii strains. Further PCR studies included the analysis of the occurrence of genetic determinants associated with efflux pump, insertion sequence and biofilm formation. The relationship between bacterial strains was assayed using 6 primers in RAPD-PCR method. Results: Detection of the blaOXA-51-like gene confirmed that the strains belong to the A. baumannii species. In the multiplex-PCR, the presence of the blaOXA-23-like and blaOXA-40-like genes was detected in 3 (10%) and 27 (90%) isolates, respectively. Moreover, some strains showed the coexistence of the blaOXA-51-like and blaOXA-23-like genes (10%, n=3) or blaOXA-51-like and blaOXA-40-like (90%, n=27). In the group of analysed strains the presence of the efflux pump gene (adeA) and the insertion sequence ISAba1 were demonstrated in all tested isolates. Biofilm-related genes (abaI, csuE) were found in 100% and 97% (n=29) tested strains adequately. The RAPD-PCR studies revealed the presence of 10 unrelated genotypes. Conclusions: The obtained results suggest that the phenomenon of multi-drug resistance in the studied A. baumannii strains could be attributed to the occurrence of CHDL oxacillinases, AdeABC efflux pump, insertion sequence ISAba1 and the biofilm formation.

Molecular characterisation of the first New Delhi metallo-β-lactamase 1-producing Acinetobacter baumannii from Tanzania.

BackgroundWe aimed to characterise the genetic determinants and context of two meropenem-resistant clinical isolates of Acinetobacter baumannii isolated from children hospitalised with bloodstream infections in Dar es Salaam, Tanzania.MethodsAntimicrobial susceptibility was determined by disc diffusion E-test and broth microdilution. Genomes were completed using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads and characterisation of the genetic context of resistance genes, multi-locus sequence types (STs) and phylogenetic analysis was determined bioinformatically.ResultsTwelve A. baumannii were isolated from 2226 blood cultures, two of which were meropenem-resistant. The two meropenem-resistant isolates, belonging to distinct STs, ST374 and ST239, were found to harbour blaNDM-1, which was chromosomally located in isolate DT0544 and plasmid-located in isolate DT01139. The genetic environment of blaNDM-1 shows the association of insertion sequence ISAba125 with blaNDM-1 in both isolates. Both isolates also harboured genes conferring resistance to other β-lactams, aminoglycosides and cotrimoxazole.ConclusionsThis is the first report of New Delhi metallo-β-lactamase-producing isolates of A. baumannii from Tanzania. The genetic context of blaNDM-1 provides further evidence of the importance of ISAba125 in the spread of blaNDM-1 in A. baumannii. Local surveillance should be strengthened to keep clinicians updated on the incidence of these and other multidrug-resistant and difficult-to-treat bacteria.

Antibiotic resistance pattern of Acinetobacter baumannii from burns patients: increase in prevalence of blaOXA-24-like and blaOXA-58-like genes

Notwithstanding the increased prevalence of Acinetobacter baumannii drug-resistant isolates, treatment options are progressively limiting. This study aims to provide a recent report on antibiotic susceptibility in burn wound isolates of A. baumannii, and the importance of OXA beta-lactamases in carbapenem resistance. The susceptibility levels to different antimicrobial categories were determined among 84 A. baumannii isolates from burn wound infection between 2016 and 2018. Multiplex PCR was used to detect OXA beta-lactamases genes, including bla OXA-51, bla OXA-23, bla OXA-24 and bla OXA-58. ISAba-1 association with bla OXA-51, bla OXA-23 and bla OXA-58 was detected by PCR mapping. All the isolates were determined as multidrug-resistant (MDR) and 69% as extensively drug-resistant (XDR). Different carbapenems MIC ranges (MIC50 and MIC90) were observed among the isolates harboring bla OXA-like genes and isolates with the OXA-24-like enzyme showed higher carbapenems MIC ranges. The prevalence of bla OXA-51-like, bla OXA-23-like, bla OXA-24-like and bla OXA-58-like were 100%, 53.57%, 41.66% and 30.95%, respectively. ISAba-1 insertion sequence was found to be upstream to bla OXA-23-like and bla OXA-58-like genes in 23 out of 45 (71.1%) bla OXA-23-like-positive and 4 out of 23 (15.3) bla OXA-58-like-positive isolates, respectively. Resistance to carbapenems as the last resort for treatment of A. baumannii infections is growing. This study, for the first time in Iran, has observed the increased frequency of bla OXA-24-like and bla OXA-58-like genes and found an association between ISAba-1 and bla OXA-58-like gene, which signifies the possible risk of increased diversity in OXA beta-lactamases and growth in carbapenem resistance.

Prevalence and molecular analysis of multidrug-resistant Acinetobacter baumannii in the extra-hospital environment in Mthatha, South Africa

IntroductionThe presence of Acinetobacter baumannii outside hospitals remains unclear. This study aimed to determine the prevalence of multidrug-resistance (MDR) A. baumannii in the extra-hospital environment in Mthatha, South Africa and to investigate the frequency of carbapenemase-encoding genes. Material and MethodsFrom August 2016 to July 2017 a total of 598 abattoir samples and 689 aquatic samples were collected and analyzed presumptively by cultural methods for the presence of A. baumannii using CHROMagar™ Acinetobacter medium. Species identification was performed by autoSCAN-4 (Dade Behring Inc., IL) and confirmed by the detection of their intrinsic blaOXA-51 gene. Confirmed MDR A. baumannii isolates were screened for the presence of carbapenemase-encoding genes, ISAba1 insertion sequence and integrase intI1. ResultsIn total, 248 (19.3%) Acinetobacter species were isolated. Acinetobacter. baumannii was detected in 183 (73.8%) of which 85 (46.4%) and 98 (53.6%) were recovered from abattoir and aquatic respectively. MDR A. baumannii was detected in 56.5% (48/85) abattoir isolates and 53.1% (52/98) aquatic isolates. Isolates showed high resistance to antimicrobials most frequently used to treat Acinetobacter infections such as piperacillin/tazobactam; abattoir (98% of isolates resistant), aquatic (94% of isolates resistant), ceftazidime (84%, 83%), ciprofloxacin (71%, 70%), amikacin (41%, 42%), imipenem (75%, 73%), and meropenem (74%, 71%). All the isolates were susceptible to tigecycline and colistin. All the isolates carried blaOXA-51-like. The blaOXA-23 was detected in 32 (66.7%) abattoir isolates and 11 (21.2%) aquatic isolates. The blaOXA-58-like was positive in 7 (14.6%) and 4 (7.7%) abattoir and aquatic isolates, respectively. Both groups of isolates lacked blaOXA-24-like, blaIMP-type, blaVIM-type, blaNDM-1,blaSIM, blaAmpC, ISAba1 and inI1. Isolates showed high level of Multiple Antibiotic Resistance Index (MARI) ranging from 0.20-0.52. ConclusionExtra-hospital sources such as abattoir and aquatic environments may be a vehicle of spread of MDR A. baumannii strains in the community and hospital settings.

Molecular epidemiology and decreased susceptibility to disinfectants in carbapenem-resistant Acinetobacter baumannii isolated from intensive care unit patients in central China

BackgroundInfection with carbapenem-resistant Acinetobacter baumannii (CRAB) is an increasing problem for critically ill patients. The srains are frequently resistant to all antibiotics and disinfectants are often used to block the spread of these bacteria, playing an important role in infection control. ObjectivesThe aim of this study was to investigate the antibiotic susceptibility, the clonal relationship, disinfectant resistance gene, β-lactamase genes and the disinfectant sensitivity of 82 A. baumannii isolates collected at a large hospital in Wuhan, China. DesignA retrospective basic study. MethodsHere we investigated 82 A. baumannii isolates from intensive care unit patients in a major teaching hospital in China for the distribution of resistance-associated genes and susceptibility to chlorine disinfectant (CLR), benzalkonium bromide (BB) and Chlorhexidine gluconate(CHG). Multi-locus sequence typing (MLST) was applied to explore their genetic evolution relationships. ResultsqacE (30.48%, 25/82) and qac△E1 (76.82%, 63/82) genes were detected in our study, while none were positive for qacA/B, qacC/D or qacG. The MIC values of CLR were 250mg/L; The MIC values ranged from 32 to 128μg/mL for BB; The MIC values ranged from 0.0019% to 0.0078% for CHG. The presence or absence of qacE gene has a significant impact(p＜0.05) on MICs of BB or CHG. All isolates harboured blaOXA-51/23 genes, and 98.78% of isolates contained the ISaba1 insertion sequence. All isolates were classified into 8 sequence types(STs) within clonal complex 92(CC92). ConclusionsThe predominant CRAB strains in our intensive care unit are blaOXA-23-containing A. baumannii of CC92. The high prevalence of qac genes and reduced susceptibility to disinfectants confirm the need for continued vigilance against nosocomial infections.

Carbapenem-resistant Acinetobacter baumannii isolates carrying blaOXA genes with upstream ISAba1: First report of a novel OXA subclass from Iran

Carbapenem-resistant Acinetobacter baumannii (CRAB) have emerged as a serious threat to public-health worldwide. This study aimed to determine the antimicrobial susceptibility of A. baumannii isolates in Iran and to investigate oxacillinase-encoding determinants and their association with insertion sequence ISAba1 in CRAB isolates. This study was performed on A. baumannii isolates recovered from patients with burn wound infections during 2013. All isolates were evaluated for antimicrobial susceptibility by the disk diffusion method. Minimum inhibitory concentrations (MICs) of five antibiotics (imipenem, meropenem, polymyxin B, colistin and tigecycline) were determined for all CRAB isolates. PCR was performed to determine the distribution of blaOXA determinants and ISAba1 insertion upstream of each corresponding gene in the CRAB isolates. A total of 65 A. baumannii isolates were recovered during the 1-year period, with CRAB accounting for 63 (96.9%) of isolates. Polymyxin B, colistin and tigecycline were the most effective agents against CRAB isolates, with susceptibility rates of 100%, 87.3% and 65.1%, respectively. The proportion of CRAB isolates carrying oxacillinase determinants was as follow: blaOXA-51-like, 100%; blaOXA-23-like, 74.6%; blaOXA-24/40-like, 47.6%; and blaOXA-235-like, 12.7%. ISAba1, ISAba1-blaOXA-23-like and ISAba1-blaOXA-51-like were detected in 100%, 41.3% and 1.6% of CRAB isolates, respectively. Co-occurrence of blaOXA determinants or inserted ISAba1 upstream of the corresponding genes was associated with increased carbapenem MICs (≥128μg/mL). The emergence of high-level CRAB with blaOXA and ISAba1-blaOXA family in burn patients is a matter of increasing clinical concern, emphasising the need for infection control efforts to limit such problematic bacteria.

A high mortality rate associated with multidrug-resistant Acinetobacter baumannii ST79 and ST25 carrying OXA-23 in a Brazilian intensive care unit.

The global spread of carbapenem-resistant Acinetobacter baumannii (A. baumannii) strains has restricted the therapeutic options available to treat infections due to this pathogen. Understanding the prevalence of such infections and the underlying genetic mechanisms of resistance may help in the implementation of adequate measures to control and prevent acquisition of nosocomial infections, especially in an intensive care unit setting. This study describes the molecular characteristics and risk factors associated with OXA-23-producing A. baumannii infections. A case-control study was undertaken from September/2013 to April/2015. Acquisition of OXA-23-producing A. baumannii was found to be associated with the use of nasogastric tubes, haemodialysis, and the use of cephalosporins. These isolates were only susceptible to amikacin, gentamicin, tigecycline, and colistin, and contained the ISAba1 insertion sequence upstream ofblaOXA-23 and blaOXA-51 genes. Twenty-six OXA-23-producing A. baumannii strains belonged to the ST79 (CC79) clonal group,and patients infected or colonised by these isolates had a higher mortality rate (34.6%). In conclusion, this study showed a dissemination of OXA-23-producing A. baumannii strains that was associated with several healthcare-related risk factors and high mortality rates among intensive care unit patients.

Co-production of AmpC and extended spectrum beta-lactamases in cephalosporin-resistant Acinetobacter baumannii in Egypt.

Acinetobacter baumannii is an opportunistic pathogen that has been held responsible for a lot of infections worldwide. Infections caused by this pathogen are difficult to control because of the widespread of antimicrobial resistance mechanisms. The aim of the present study is to assess the prevalence of extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases among isolates of A. baumannii collected from different clinical sources in Mansoura University Hospitals, Egypt. Antimicrobial susceptibility testing has demonstrated elevated resistance level to β-lactams, quinolones and aminoglycosides. All isolates were sensitive to colistin and polymyxin B. ESBL activity was detected in 86% of the isolates. Among the tested ESBL encoding genes, blaTEM gene was the most prevalent gene as it was detected in 52% of the isolates. While blaPER, blaSHV and blaVEB were detected in 12%, 4%, and 2%, respectively. AmpC activity and blaADC gene were detected in 90% of the tested isolates. Insertion sequence ISAba1 was located 9bp upstream of blaADC gene in 88.9% of the ADC-expressing isolates providing a potent promoter activity for its expression. To our knowledge this is the first report of loss of intrinsic ADC activity, in 10% of the tested isolates, as a result of insertional inactivation by an element belonging to IS5 family transposase. Co-expression of both ESBLs and AmpC β-lactamases was detected in 78% of the isolates. The study demonstrates high prevalence of resistance to β-lactam antibiotics through ESBLs and AmpC β-lactamases production among A. baumannii clinical isolates. Prevalence of β-lactamases should be detected routinely and reported in hospitals to avoid inappropriate use of antibiotics and therapeutic failure.

ISAba1 and Tn6168 acquisition by natural transformation leads to third-generation cephalosporins resistance in Acinetobacter baumannii

Acinetobacter baumannii has intrinsic beta-lactamase genes, namely ampC and blaOXA-51-like, which are only strongly expressed when the ISAba1 insertion sequence is upstream the 5′ end of the genes. A second ampC gene has also been identified in some clinical A. baumannii strains. The increased expression of these genes leads to resistance to beta-lactams, including third-generation cephalosporins and/or carbapenems. The aim of this work was to assess the involvement of natural transformation in the transfer of chromosomal ampC-associated mobile elements, and related changes in the resistance profile of recipient cells. Natural transformation assays with the naturally competent A. baumannii A118 clinical isolate as recipient cell and the multidrug resistant A. baumannii Ab51 clinical isolate as the source of donor DNA produced transformants. All tested transformants showed integration of the ISAba1 close to the ampC gene. In two transformants, the ISAba1 was acquired by transposition and inserted between the usual folE and the ampC genes. The remaining transformants acquired the ISAba1 adjacent to a second ampC gene, as part of Tn6168, likely by homologous recombination. Our study demonstrates that natural transformation can contribute to the widespread of beta-lactams resistance, and acquisition of non-resistant determinants can lead to changes in the susceptibility profile of A. baumannii strains.

Carbapenemase Production of Clinical Isolates Acinetobacter baumannii and Pseudomonas aeruginosa from a Bulgarian University Hospital.

Production of Bla OXA-23, OXA-24, OXA-58 and hyperexpression of OXA-51 due to ISAba1 insertion sequence are the leading causes of carbapenem resistance in Acinetobacter baumannii. The loss of OprD transmembrane protein and the overexpression of some effl ux pumps are considered to be the main factors for carbapenem resistance in Pseudomonas aeruginosa whereas metallo-enzymes' production has a secondary role. Тo examine the carbapenem resistance due to carbapenemase production among clinically signifi cant Gram-negative non-fermenters from St George University hospital, Plovdiv: A. baumannii and P. aeruginosa. Forty three A. baumannii and 43 P. aeruginosa isolates, resistant or with intermediate resistance to imipenem and/or meropenem were included in the study. They were collected from patients admitted in 14 various hospital wards between 2010 and 2014. Both phenotypic and genetic methods were used for identifi cation and antimicrobial susceptibility testing. All A. baumannii demonstrated carbapenemase production determined by a modifi ed Hodge test whereas P. aeruginosa isolates did not show this phenomenon. OXA-23 genes were determined in 97.7% (42 out of 43) of A. baumannii isolates indistinguishable from the sequence of the classical ARI-1 gene. OXA-24, OXA-58 and overexpression of OXA-51 were not registered in any of the isolates. All P. aeruginosa were negative for blaVIM and blaIMP genes. The leading cause of carbapenem resistance in A. baumannii isolates from our hospital is the carbapenemase production due to the expression of OXA- 23 gene, whereas in P. aeruginosa - the loss of transmembrane OprD protein and the effl ux pumps' hyperexpression are suspected to be the main mechanisms.

Polyclonal Distribution of blaOXA-23 Gene Among Acinetobacter baumannii Isolated from Intensive Care Unit Patients in Tehran; Pulsed-Field Gel Electrophoresis Analysis

Background: Despite a relatively low virulence of Acinetobacter baumannii isolates, emerging multidrug-resistant (MDR) strains to pose a formidable threat to patients, particularly in ill patients in intensive care unit (ICU). Objectives: The aim of the present study was to determine the genetic relatedness and antimicrobial susceptibility patterns in the endemic clones of A. baumannii isolated from patients in the ICU. Methods: Fifty-five non-repetitive A. baumannii isolates were examined for antimicrobial susceptibility, oxacillinase genes, class 1 integrons and genetic relationships by PFGE. Results: Antibiotic susceptibility testing showed that 21.8%) 12 isolates) were resistant to all tested antibiotics. Resistant to carbapenems were up to 85%. OXA-23, OXA-24 and OXA-58 genes were detected in 81.81%, 16.36% and 1.81% isolates respectively. The ISAba1 element upstream of blaOXA-51 was detected in 18 (32%) isolates, and 22 (40%) isolates had an ISAba1 insertion sequence upstream of the blaOXA-23. Integron class 1 was detected in 25 (55.5%) OXA-23 carrying isolates, 2 (22.2%) in OXA-24 positive isolates and in one OXA-58 carrying isolate. PFGE analysis resolved 50 distinct pulsotypes, Most of the isolates were scattered throughout across the dendrogram and a few grouped as clusters. Conclusions: No significant association has been found between the pulsotype of each isolate and MDR patterns and the presence of carbapenemase genes, however, highly resistant blaOXA-23 gene carrying endemic clones of A. baumannii disseminated in the ICU of two hospitals. Therefore, active surveillance and health policies are urgently needed for the detection and control the dissemination of such organisms.