Carbapenem Resistance Determinants Research Articles

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant K. aerogenes (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Carbapenemase-encoding genes and drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICE) which has been associated with pathogenic Klebsiella pneumoniae lineages (ICEKp10). Comparative genomics with global K. aerogenes genomes showed our outbreak strains to group closely with global sequence type 4 (ST4) strains, which, along with ST93, likely represent dominant K. aerogenes lineages associated with human infections. For poorly characterized pathogens, scaling analyses to include sequenced genomes from public databases offer the opportunity to identify emerging trends and dominant clones associated with specific attributes, syndromes, and geographical locations.

Acinetobacter baumannii analysis by core genome multi-locus sequence typing in two hospitals in Bolivia: endemicity of international clone 7 isolates (CC25)

In total, 95 Acinetobacter baumannii isolates recovered from patients from two hospitals in Cochabamba, Bolivia were studied. The presence of class D and B β-lactamases was investigated using polymerase chain reaction, and antimicrobial susceptibility testing was performed by agar dilution and broth microdilution. The resistance rate to carbapenems was 53.7%. All carbapenem-resistant A. baumannii (CRAb, n=51) and four carbapenem-susceptible isolates were further analysed by whole-genome sequencing. The resulting genome assemblies were used to identify the acquired resistome, and core genome multi-locus sequence typing (cgMLST) was used to determine their molecular epidemiology. All but one of the CRAb isolates (n=50) belonged to international clone (IC) 7 and they clustered into five sequence types; on cgMLST, they were found to be separated by ≥40 alleles. All CRAb isolates carried blaOXA-23 on transposon Tn2008. Metallo-β-lactamases were not detected. These data show that dissemination of several IC7 A. baumannii clones harbouring the carbapenem resistance determinant blaOXA-23 is occurring in these two hospitals in Cochambamba.

Molecular epidemiology of colistin-resistant Pseudomonas aeruginosa producing NDM-1 from hospitalized patients in Iran.

Objective(s):Resistance to carbapenems is the principal reason for the continuing utilization of colistin as a last resort choice for treating the infections resulted from multidrug carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates. The assessment of antimicrobial resistance pattern, the prevalence of carbapenem-resistance determinants, and molecular epidemiology of colistin-resistant isolates among CRPA strains were the aims of the present research. Materials and Methods:The current cross-sectional research was conducted on 269 CRPA isolates collected from various clinical samples from 2013 to 2016. After performing identification tests, disk diffusion as well as MIC methods were used for testing sensitivity to the antibiotics. Modified Hodge Test (MHT) was utilized to produce carbapenemase. PCR technique identified beta-lactamase classes A, B, and D genes. Results:In total, from 269 CRPA, five isolates (1.3%) were resistant to colistin. It was found that blaNDM-1, blaIMP-1, blaVIM-2, and blaOXA-10 genes were present in 40%, 40%, 20%, and 100% of colistin-resistant isolates, respectively. DLST type 25-11 is a significant cluster of colistin-resistant P. aeruginosa isolates. Conclusion:The appearance of colistin-resistant isolates in CRPA carrying blaNDM-1 with multiple carbapenem-resistant genes shows the great problem in the treatment of P. aeruginosa infections.

Phenotypic and molecular characterization of Acinetobacter baumannii isolates causing lower respiratory infections among ICU patients

BackgroundMulti-drug resistant Acinetobacter baumannii has emerged as important nosocomial pathogen associated with various infections including lower respiratory tract. Limited therapeutic options contribute to increased morbidity and mortality. Acinetobacter baumannii has the ability to persist in the environment for prolonged periods. Breach in infection control practices increases the chances of cross transmission between patients and inter/intraspecies transmission of resistance elements. The present prospective work was conducted among patients with lower respiratory tract infections (LRTI) in the intensive care unit (ICU) to study the etiology with special reference to Acinetobacter baumannii and the role of immediate patient environment in the ICU as possible source of infection. Acinetobacter baumannii were characterized for antimicrobial susceptibility, mechanism of carbapenem resistance and virulence determinants. Molecular typing of the clinical and environmental isolates was undertaken to study the probable modes of transmission. Materials and methodsAppropriate respiratory samples from 107 patients with LRTI admitted to ICU during September 2016 to March 2017 were studied for likely bacterial pathogens. Environmental samples (n = 71) were also screened. All the samples were processed using conventional microbiological methods. Consecutive Acinetobacter spp. isolated from clinical and environmental (health care workers and environment from ICU) samples were included in the study. Antimicrobial susceptibility was performed as per CLSI guidelines. Carbapenem resistance, mediated by carbapenemase genes (blaOXA-23-like,blaOXA-24-like,blaOXA-58-like and blaNDM-1) were studied by PCR. Biofilm forming ability was tested phenotypically using microtitre plate method. Pulse Field Gel Electrophoresis (PFGE) was used to study clonality of the clinical and environmental isolates. ResultsThe prevalence of Acinetobacter baumannii was 26.2% (28/107) and 11.26% (8/71) among patients with LRTI and environmental samples respectively. The carbapenem resistance was high, 96.42% (27/28) and 87.5% (7/8) in clinical and environmental isolates respectively. The most common carbapenemase associated with resistance was blaOXA-23-like gene followed by blaNDM-1 among both the clinical and environmental isolates. All isolates were sensitive to colistin (MIC ≤ 1 μg/ml). Biofilm production was observed among all clinical (n = 28) and 87.5% (7/8) of the environmental isolates. Line listing of the cases suggests the occurrence of infections throughout the study period with no significant clustering. On PFGE, 12 clusters were observed and 16/36 isolates were present in one single cluster that included both clinical and environmental isolates which were either carbapenem resistant or sensitive. DiscussionCarbapenem resistant Acinetobacter baumannii (CRAB) is an important cause of LRTI in the ICU. PFGE suggests spread of carbapenem resistant isolates via cross transmission among patients and the environment. The detection of blaNDM-1 gene among Acinetobacter baumannii and existence of carbapenem resistant and sensitive isolates within the same clones suggests horizontal transmission of resistant genes among various bacterial species. The ability of Acinetobacter baumannii to form biofilms may contribute to its persistence in the environment. This along with breach in infection control practices are the likely factors contributing to this transmission. This information can be used to strengthen and monitor infection control (IC) and the hospital cleaning and disinfection practices to prevent spread of resistant organisms within the ICU. Colistin remains drug of choice for management of CRAB.

Molecular Epidemiology and Drug Resistance Pattern of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Iran.

The emergence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and their involvement in several nosocomial outbreaks are of high concern. This study was conducted to investigate the genetic relatedness and molecular determinants of carbapenem resistance in 100 CRKP isolates. Susceptibility to carbapenems as well as other antibiotics was determined by using disk diffusion method. The Modified Hodge test was performed for detection of carbapenemase production. The minimum inhibitory concentrations of selected antibiotics were determined by broth microdilution method. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR, and clonal relatedness of CRKP isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. blaOXA-48 was the most frequent carbapenemase gene (72%), followed by blaNDM (31%). None of the isolates harbored blaKPC and blaVIM genes. PFGE separated the majority of isolates into 10 clusters, including the major clusters A and B, carrying blaOXA-48, and clusters C and D, carrying blaNDM, and 4 isolates had a unique PFGE pattern. An increased rate of colistin resistance (50%) was detected among the isolates. Tigecycline was found to be the most active agent against CRKP isolates. Our results revealed that high prevalence of blaOXA-48 and blaNDM carbapenamses and resistance to colistin are alarming threats, necessitating an immediate action to prevent the spread of carbapenem-colistin-resistant K. pneumoniae isolates in Iran.

The epidemiological impact and significance of carbapenem resistance in Pseudomonas aeruginosa bloodstream infections: a matched case-case-control analysis.

A case-case-control investigation (N = 255 patients) explored the epidemiology of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Recent exposure to carbapenems and a rapidly fatal condition should prompt practitioners to shorten delays in initiating appropriate therapy, which can adversely impact CRPA outcomes, as opposed to the isolated impact of the carbapenem resistance determinant.

Genomic and Geographic Context for the Evolution of High-Risk Carbapenem-Resistant Enterobacter cloacae Complex Clones ST171 and ST78.

ABSTRACTRecent reports have established the escalating threat of carbapenem-resistant Enterobacter cloacae complex (CREC). Here, we demonstrate that CREC has evolved as a highly antibiotic-resistant rather than highly virulent nosocomial pathogen. Applying genomics and Bayesian phylogenetic analyses to a 7-year collection of CREC isolates from a northern Manhattan hospital system and to a large set of publicly available, geographically diverse genomes, we demonstrate clonal spread of a single clone, ST171. We estimate that two major clades of epidemic ST171 diverged prior to 1962, subsequently spreading in parallel from the Northeastern to the Mid-Atlantic and Midwestern United States and demonstrating links to international sites. Acquisition of carbapenem and fluoroquinolone resistance determinants by both clades preceded widespread use of these drugs in the mid-1980s, suggesting that antibiotic pressure contributed substantially to its spread. Despite a unique mobile repertoire, ST171 isolates showed decreased virulence in vitro. While a second clone, ST78, substantially contributed to the emergence of CREC, it encompasses diverse carbapenemase-harboring plasmids, including a potentially hypertransmissible IncN plasmid, also present in other sequence types. Rather than heightened virulence, CREC demonstrates lineage-specific, multifactorial adaptations to nosocomial environments coupled with a unique potential to acquire and disseminate carbapenem resistance genes. These findings indicate a need for robust surveillance efforts that are attentive to the potential for local and international spread of high-risk CREC clones.

Genomic Investigation of a Putative Endoscope-Associated Carbapenem-Resistant Enterobacter cloacae Outbreak Reveals a Wide Diversity of Circulating Strains and Resistance Mutations.

A genomic epidemiologic investigation of a putative carbapenem-resistant Enterobacter cloacae outbreak revealed few plausible instances of nosocomial transmission, highlighting instead the frequent importation of E. cloacae into our hospital. Searching for genetic determinants of carbapenem resistance demonstrated that most resistance is due to convergent mutations in phylogenetically diverse E. cloacae.

High incidence of pandrug-resistant Acinetobacter baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial.

ObjectivesTo investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial.Methods A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database.ResultsOf 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates.ConclusionsCarbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii.

Occurrence of Carbapenemase-Producing Enterobacteriaceae Isolates in the Wildlife: First Report of OXA-48 in Wild Boars in Algeria.

The aim of the present study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from wild boars and Barbary macaques in Algeria. Fecal samples were collected from wild boars (n = 168) and Barbary macaques (n = 212), in Bejaia, Algeria, between September 2014 and April 2016. The isolates were identified and antimicrobial susceptibility was determined. Carbapenem resistance determinants were studied using PCR and sequencing, while clonal relatedness was performed using multilocus sequence typing (MLST). PCR was used to investigate certain virulence genes. Three CPE isolates from three different samples (1.8%) recovered from wild boars were identified as Escherichia coli (two isolates) and Klebsiella pneumoniae (one isolate). These isolates were resistant to amoxicillin, amoxicillin-clavulanate, tobramycin, ertapenem, and meropenem. The results of PCR and sequencing analysis showed that all three isolates produced the OXA-48 enzyme. The MLST showed that the two E. coli isolates were assigned to the same sequence type, ST635, and belonged to phylogroup A, whereas K. pneumoniae strain belonged to ST13. The K. pneumoniae strain was positive for multiple virulence factors, whereas no virulence determinants were found in E. coli isolates. This is the first report of OXA-48-producing Enterobacteriaceae in wild animals from Algeria and Africa.

Multidrug Resistance Mechanisms of Carbapenem Resistant Klebsiella pneumoniae Strains Isolated in Chongqing, China.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics.MethodsPCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays.ResultsSeventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum β-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates.ConclusionsA high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of blaNDM-1 carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring blaNDM-1, blaKPC-2, blaCTX-M, blaSHV, acc(6′)-Ib, rmtB, qnrB, and acc(6′)-Ib-cr.

Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli.

Carbapenemase-producing gram-negative bacteria (CP-GNB) are an urgent and expanding public health threat. Rapid and accurate identification of these organisms facilitates infection prevention efforts in healthcare facilities. The objective of our study was to evaluate methods to detect and identify CP-GNB. We examined 189 carbapenem-resistant GNB(CR-GNB), including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex, using 3 different methods: 2 methods to screen isolates of GNB for carbapenemase production [the carbapenem inactivation method (CIM) and 2 chromogenic agars] and a molecular method (Cepheid GeneXpert Carba-R) to identify the mechanism of carbapenem resistance and the associated resistance genes (blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM). The CIM was a simple and inexpensive phenotypic screen to differentiate between CR-GNB and CP-GNB, with improved analytical performance characteristics and inter-reader correlation compared to the modified Hodge test. Both chromogenic agars evaluated (HardyCHROM CRE and chromID CARBA) were able to support growth of most of the organisms tested, including isolates possessing the blaOXA-48-like gene. However, these media had a low analytical specificity for carbapenemase production, with breakthrough of CR-GNB that did not produce a carbapenemase. The Xpert Carba-R assay was rapid and easy to perform, and demonstrated 100% positive and negative agreement for characterization of genetic determinants of carbapenem resistance. Screening by CIM followed by the Xpert Carba-R PCR is an accurate method for detecting and characterizing CP-GNB, including Enterobacteriaceae, P. aeruginosa, and A. baumannii complex.

Reduced Ertapenem Susceptibility Due to OXA-2 Production in Klebsiella pneumoniae ST258

OXA-2 is a class D β-lactamase which is primarily found in E. coli (Ec) and P. aeruginosa and confers resistance to penicillins as well as narrow-spectrum cephalosporins. However, recent reports suggest that OXA-2 also possesses carbapenem hydrolyzing activity. We report a case of K. pneumoniae that confers reduced susceptibility to ceftazidime and ertapenem due to production of OXA-2. K. pneumoniae (Kp) strain YDC787 was identified from the BAL culture of an inpatient in December 2016. The strain was initially reported as intermediate to ertapenem. MICs and carbapenemase production were confirmed by Etest and modified Hodge test (MHT) respectively. Ec TOP10 was transformed with purified plasmids followed by selection with ampicillin (TOP10 [pYDC787]), and the carbapenem resistance determinant was cloned into vector pBCSK using partial Sau3AI digestion (TOP10 [pOXA-2]). Kp YDC787, Ec TOP10 (pYDC787), and Ec TOP10 (pOXA-2) all showed reduced susceptibility to ertapenem and ceftazidime, but not meropenem, imipenem, or cefepime. All isolates were positive for carbapenemase production by MHT. Sanger sequencing of pOXA-2 revealed 2,910-bp partial class 1 integron containing DaacA4-blaOXA-2-qacED1-Dsul1. The presence of blaOXA-2 was confirmed by PCR in Kp YDC787 and Ec TOP10 (pYDC787). These findings confirm that OXA-2 functions as carbapenemase and cephalosporinase, significantly reducing susceptibility to ertapenem and ceftazidime, but not to meropenem, imipenem, or cefepime. All authors: No reported disclosures.

Effect of single-dose carbapenem exposure on transcriptional expression of blaNDM-1 and mexA in Pseudomonas aeruginosa.

The therapeutic option of a carbapenem antibiotic is compromised in Pseudomonas aeruginosa owing both to acquired and intrinsic resistance mechanisms. In recent years, New Delhi metallo-β-lactamase has been the focus as a predominant carbapenem resistance determinant. However, it is unclear which of the mechanisms might be adopted by a P. aeruginosa strain possessing both blaNDM-1 and an overexpressed MexAB-OprM system during carbapenem therapy. This study investigated the interplay of both mechanisms in clinical isolates of P. aeruginosa when exposed to meropenem. Five strains were used: (i) strain overexpressing MexAB-OprM but with no blaNDM-1; (ii) strain harbouring blaNDM-1 but expressing MexAB-OprM at basal level; (iii) strain possessing blaNDM-1 and overexpressing MexAB-OprM; (iv) P. aeruginosa PAO1; and (v) P. aeruginosa K2733-PAO1 (ΔMexAB-OprMΔMexCD-OprJΔMexEF-OprNΔMexXY-OprM) into which blaNDM-1 was cloned. Strains were incubated in Luria-Bertani broth with and without 1μg/mL meropenem. Total RNA was isolated at 45-min intervals and was immediately reverse transcribed to cDNA. This was repeated for 6h. Quantitative real-time PCR was performed for both resistance mechanisms. Meropenem exposure did not significantly elevate transcription of either the blaNDM-1 or mexA gene. However, an interesting finding was that upon single-dose exposure to carbapenem, the efflux pump system played a major role in bacterial survival compared with NDM-1. This study gives an insight into the bacterial response to carbapenem antibiotic when two different resistance mechanisms coexist. This type of study would be helpful in designing future antimicrobials.

Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt.

This study investigated the occurrence of carbapenem-resistant Klebsiella pneumoniae strains in broiler chickens, drinking water and humans working in contact with chickens and identified the carbapenem resistance determinants among isolates from different sources. Internal organs and droppings were collected from 100 broilers with signs of respiratory disease at five broiler farms in Egypt. Additionally, 20 drinking water samples and 49 faecal samples from workers and veterinarians working at these farms were included. Following culture on MacConkey agar, suspected K. pneumoniae colonies were identified by phenotypic testing. Susceptibility to carbapenems was tested in confirmed K. pneumoniae isolates by disk diffusion. Carbapenem-resistant isolates were subjected to PCR for detection of carbapenemase-encoding genes (blaKPC, blaOXA-48 and blaNDM). K. pneumoniae was isolated from 35% of broilers and 25% of water samples. Of the 35 poultry isolates, 15 were carbapenem-resistant; all of them were blaNDM-positive, including 11 isolates harbouring blaKPC, blaOXA-48 and blaNDM and 4 containing either blaKPC and blaNDM (n=3) or blaOXA-48 and blaNDM (n=1). Similarly, three of five K. pneumoniae isolates from drinking water were positive for blaKPC and blaNDM (n=1) or for all three genes (n=2). Interestingly, 56% of K. pneumoniae from humans displayed carbapenem resistance; all of them were positive for the three carbapenemase genes. Carbapenemase-producing K. pneumoniae occurred at relatively high frequency among broilers, drinking water and workers at poultry farms in Egypt. Additional work is needed to confirm transmission between poultry and humans and to elucidate the direction and mechanism of transmission.

Early detection of metallo-β-lactamase NDM-1- and OXA-23 carbapenemase-producing Acinetobacter baumannii in Libyan hospitals

Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The aim of this study was to characterise the molecular support of carbapenem-resistant A. baumannii clinical isolates recovered from two Libyan hospitals. Bacterial isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was performed using disk diffusion and Etest methods, and carbapenem resistance determinants were studied by PCR amplification and sequencing. Multilocus sequence typing (MLST) was performed for typing of the isolates. All 36 imipenem-resistant isolates tested were identified as A. baumannii. The blaOXA-23 gene was detected in 29 strains (80.6%). The metallo-β-lactamase blaNDM-1 gene was detected in eight isolates (22.2%), showing dissemination of multidrug-resistant (MDR) A. baumannii in Tripoli Medical Center and Burn and Plastic Surgery Hospital in Libya, including one isolate that co-expressed the blaOXA-23 gene. MLST revealed several sequence types (STs). Imipenem-resistant A. baumannii ST2 was the predominant clone (16/36; 44.4%). This study shows that NDM-1 and OXA-23 contribute to antibiotic resistance in Libyan hospitals and represents the first incidence of the association of these two carbapenemases in an autochthonous MDR A. baumannii isolated from patients in Libya, indicating that there is a longstanding infection control problem in these hospitals.

Clinical and Epidemiological Significance of Carbapenem Resistance in Acinetobacter baumannii Infections.

Carbapenems are considered the treatment of choice for Acinetobacter baumannii infections. Many facilities implement preventive measures toward only carbapenem-resistant A. baumannii (CRAB). However, the independent role of the carbapenem resistance determinant on patient outcomes remains controversial. In a 6-year analysis of adults with A. baumannii bloodstream infection (BSI), the outcomes of 149 CRAB isolates were compared to those of 91 patients with carbapenem-susceptible A. baumannii In bivariable analyses, CRAB BSIs were significantly associated with worse outcomes and with a delay in the initiation of appropriate antimicrobial therapy (DAAT). However, in multivariable analyses, carbapenem resistance status was no longer associated with poor outcomes, while DAAT remained an independent predictor. The epidemiological significance of A. baumannii should not be determined by its resistance to carbapenems.

Characterization of NDM-1- and OXA-23-producing Acinetobacter baumannii isolates from inanimate surfaces in a hospital environment in Algeria

Investigation of several outbreaks of multidrug-resistant Acinetobacter baumannii infection has demonstrated that contamination of the inanimate hospital environment could be implicated in the spread of these multidrug-resistant strains. To investigate the occurrence of carbapenem-resistant A. baumannii on inanimate surfaces and possible dissemination in the hospital environment in Algeria as a potential source of infection in humans. A. baumannii strains were isolated from the hospital environment and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined using disc diffusion and E-test methods. Carbapenemase activity was detected using microbiological tests, including modified Hodge test, modified Carba NP test, and EDTA test. Carbapenem resistance determinants were studied by polymerase chain reaction (PCR) and sequencing. Clonal relatedness was determined using multi-locus sequence typing (MLST). A total of 67 A. baumannii isolates were obtained from 868 environmental samples and identified by MALDI-TOF MS. Among them, 61 isolates were resistant to imipenem with minimum inhibitory concentration >32 μg/mL and positive by the modified Hodge test and modified Carba NP test. In addition, the activity of carbapenemase was inhibited by EDTA in 32 strains. PCR and sequencing showed the presence of blaOXA-23 gene in 29 strains, and the blaNDM-1 gene in 32 isolates. MLST demonstrated the presence of five types of ST (ST19, ST2, ST85, ST98, and ST115). Our study demonstrated the dissemination of carbapenemase-producing A. baumannii strains recovered from inanimate surfaces in a hospital environment, surrounding patients, healthcare workers and visitors, in Algeria as a potential source for nosocomial infection.

Genetic Contexts of blaNDM-1 in Patients Carrying Multiple NDM-Producing Strains.

The carbapenem resistance determinant blaNDM-1 has been found in various Gram-negative bacteria and upon different plasmid replicon types (Inc). Here, we present four patients within two hospitals in Pakistan harboring between two and four NDM-1-producing Gram-negative bacilli of different species coresident in their stool samples. We characterize the blaNDM-1 genetic contexts of these 11 NDM-1-producing Gram-negative bacilli in addition to other antimicrobial resistance mechanisms, plasmid replicon profiles, and sequence types (STs) in order to understand the underlying acquisition mechanisms of carbapenem resistance within these bacteria. Two common plasmid types (IncN2 and IncA/C) were identified to carry blaNDM-1 among the six different bacterial species isolated from the four patients. Two of these strains were novel Citrobacter freundii ST 20 and ST 21. The same IncN2-type blaNDM-1 genetic context was found in all four patients and within four different species. The IncA/C-type blaNDM-1 genetic context was found in two different species and in two of the four patients. Combining genetic context characterization with other molecular epidemiology methods, we were able to establish the molecular epidemiological links between genetically unrelated bacterial species by linking their acquisition of an IncN2 or IncA/C plasmid carrying blaNDM-1 for carbapenem resistance. By combining plasmid characterization and in-depth genetic context assessment, this analysis highlights the importance of plasmids in antimicrobial resistance. It also provides a novel approach for investigating the underlying mechanisms of blaNDM-1-related spread between bacterial species and genera via plasmids.

Effective transfer of a 47 kb NDM-1-positive plasmid among Acinetobacter species.

To investigate the link between two NDM-1-positive Acinetobacter isolates from the same hospital, the plasmid profiles of the isolates were examined. These two isolates were found from a surveillance programme within 3 months from two patients without obvious physical contact or hospitalization time overlap. Antimicrobial susceptibility tests, genome sequencing of both isolates and plasmid transfer experiments were performed. A comparative study of similar plasmids was performed using BLAST analysis. The antimicrobial susceptibility of the isolates (Acinetobacter soli M131 and Acinetobacter pittii MS32) and their Escherichia coli transconjugants revealed a conjugative plasmid that carried the carbapenem resistance determinant. Eleven plasmids were observed in M131 and three in MS32. Each isolate shared an identical plasmid that carried the blaNDM-1 gene. This 47 271 bp plasmid harbours a conserved blaNDM-1-containing region that is flanked by ISAba125 and ISAba11 elements, and also contains a Ti-type conjugative operon. The plasmid is nearly identical in sequence to those of Acinetobacter isolates from China. In contrast to the mobilization of the blaNDM-1 sequence in Enterobacteriaceae, which is mainly by transposition, this plasmid moves as a whole among Acinetobacter species. Consistently, this plasmid was found to transfer effectively by in vitro conjugation to several Acinetobacter species. The clinical and laboratory findings suggest that Acinetobacter species may serve as a reservoir of this blaNDM-1 plasmid. Our study demonstrates the potential of applying genome sequencing to the surveillance of antimicrobial-resistant bacteria.