Carbapenemase-producing Gram-negative Bacilli Research Articles

The epidemiology of multidrug-resistant organisms in persons diagnosed with cancer in Norway, 2008–2018: expanding surveillance using existing laboratory and register data

Surveillance has revealed an increase of multidrug-resistant organisms (MDROs), even in low-prevalent settings such as Norway. MDROs pose a particular threat to at-risk populations, including persons with cancer. It is necessary to include such populations in future infection surveillance. By combining existing data sources, we aimed to describe the epidemiology of MDROs in persons diagnosed with cancer in Norway from 2008 to 2018. A cohort was established using data from the Cancer Registry of Norway, which was then linked to notifications of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin- and/or linezolid-resistant enterococci (V/LRE), and carbapenemase-producing Gram-negative bacilli (CP-GNB) from the Norwegian Surveillance System for Communicable Diseases, and laboratory data on third-generation cephalosporin-resistant Enterobacterales (3GCR-E) from Oslo University Hospital (OUH). We described the incidence of MDROs and resistance proportion in Enterobacterales from 6 months prior to the person’s first cancer diagnosis and up to 3 years after. The cohort included 322,005 persons, of which 0.3% (878) were diagnosed with notifiable MDROs. Peak incidence rates per 100,000 person-years were 60.9 for MRSA, 97.2 for V/LRE, and 6.8 for CP-GNB. The proportion of 3GCR-E in Enterobacterales in blood or urine cultures at OUH was 6% (746/12,534). Despite overall low MDRO incidence, there was an unfavourable trend in the incidence and resistance proportion of Gram-negative bacteria. To address this, there is a need for effective infection control and surveillance. Our study demonstrated the feasibility of expanding the surveillance of MDROs and at-risk populations through the linkage of existing laboratory and register data.

Rapid AST: Possibility of inferring resistance mechanisms with complex phenotypes.

The new automated systems designed for rapid performance of AST have significantly reduced the response time for susceptibility testing of microorganisms causing bacteremia and sepsis. The Accelerate Pheno® system (AAC) is one such system. Our objective for this study was to determine whether the AAC system is capable of providing an accurate susceptibility profile to infer resistance mechanisms in different carbapenemase-producing isolates when compared to the MicroScan WalkAway System (MWS). Disk diffusion method was also performed on all isolates as a reference method. Additionally, we compared the results obtained with the routine AST production system. We selected 19 isolates from the cryobank of the Microbiology department, all of which were carbapenemase-producing gram-negative bacilli. AAC was able to identify and infer the resistance of a total of 10 isolates, with an EA and CA of 84.2% for meropenem and 88.2% and 64.7% for ertapenem EA and CA, respectively. If we consider the disk diffusion technique, the CA was 57.9% and 76.5% for meropenem and ertapenem. However, in the presence of carbapenemases, AAC was not able to provide adequate MICs or infer the resistance mechanisms of the isolates accurately. Further studies with a larger number of isolates, including the new antibiotics ceftolozane/tazobactam and ceftazidime/avibactam, are needed for a more comprehensive comparison.

In Vitro Activity of Eravacycline against Carbapenemase-Producing Gram-Negative Bacilli Clinical Isolates in Central Poland.

Eravacycline is a novel antibiotic of the tetracycline class with activity against a broad spectrum of clinically significant bacteria, including multi-drug-resistant organisms. For this reason, it may be an alternative to treating critical infections of this etiology. We aimed to assess the in vitro effectiveness of eravacycline to carbapenemase-producing Gram-negative bacilli clinical isolates identified in hospitals in Łódź, Poland. We analyzed 102 strains producing KPC, MBL, OXA-48, GES, and other carbapenemases. Eravacycline susceptibility was determined following the EUCAST guidelines. The highest susceptibility was found in KPC (73%) and MBL (59%) strains. Our results confirmed in vitro the efficacy of this drug against carbapenem-resistant strains. However, eravacycline has been indicated only for treating complicated intra-abdominal infections, significantly limiting its use. This aspect should be further explored to expand the indications for using eravacycline supported by evidence-based medicine. Eravacycline is one of the drugs that could play a role in reducing the spread of multidrug-resistant microorganisms.

SG-APSIC1083: Prevalence and classification of carbapenemase-producing gram-negative bacilli at a medical center in Ho Chi Minh City, Vietnam

Objectives: The identification and classification of carbapenemases are meaningful in clinical treatment, epidemiology, and multidrug-resistant bacteria control. We sought to identify the proportion of carbapenemase-producing and carbapenemase classifications in gram-negative bacilli in our hospital. Methods: Isolates of gram-negative bacilli were extracted from sputum, blood, and urine samples in a medical center in Ho Chi Minh City. The identification of gram-negative bacilli was performed using the Phoenix M50 automated system (Becton Dickinson, Franklin Lakes, NJ). An antibiogram was conducted using the disk-diffusion method to detect meropenem-resistant gram-negative bacteria. Carbapenemase confirmation and classification of isolates resistant or intermediately resistant to meropenem were performed using the NMIC500 CPO kit on the Phoenix M50 system. Results: Among 599 isolates of gram-negative bacilli, 108 isolates were resistant or intermediately resistant to carbapenem (meropenem). Of these108 isolates, 107 (99.1%) were resistant due to the carbapenemase-producing mechanism. The proportions of resistant or intermediately resistant isolates to carbapenem were as follows: 73.8% for Acinetobacter baumannii, 26.4% for Klebsiella pneumoniae, 25.9% for Pseudomonas aeruginosa, and 2.8% for Escherichia coli. Class D carbapenemase accounted for the highest proportion, with 53 (49.5%) of 107 isolates, followed by class B with 31 isolates (29%), and class A with the lowest proportion of 2 isolates (1.9%). Also, 44.4% of Acinetobacter baumannii isolates and 74.4% of Klebsiella pneumoniae isolates produced class D carbapenemase. Conclusions: Gram-negative bacilli are resistant to carbapenem primarily due to the carbapenemase-secreting mechanism. D-class carbapenemase accounted for the highest percentage, followed by B-class type, and A-class carbapenemase in gram-negative bacilli.

DropCarba – An automated magnetic digital microfluidic platform for rapid phenotypic testing of carbapenemase-producing Gram-negative bacilli

Carbapenemase-producing Gram-negative bacilli (CPGNB) is a type of antibiotic-resistant pathogens that often lead to severe clinical consequences. Phenotypic tests, such as Carba NP and blue Carba, are able to detect the resistant mechanism and provide rapid detection of carbapenemase producers to potentially guide personalized therapy. However, these tests require relatively tedious hands-on fluidic operations, and the assay format is ill-suited for automation and parallelization for improved throughput. In this study, we report an automated magnetic digital microfluidics-based platform, known as DropCarba, for parallel CPGNB detection in droplets. It automates the entire carbapenemase testing process and eliminates the need for almost all hands-on fluidic operations, which ensures high consistency and minimizes human errors with a simple “press-and-go” operation. DropCarba was validated with a large number of bacterial isolates of various Enterobacterales species (200 strains in total with 100 CPGNB and 100 non-resistant strains) in a blinded manner, and the results agree well with the benchmark Carba NP. DropCarba, with its full automation, simple operation, reduced reagent consumption, parallelization processing, and scalable manufacturing, will greatly improve CPGNB screening and make a valuable contribution to our fight against antibiotic resistance.

The Modified Carbapenem Inactivation Method (mCIM): Highly sensitive and specific low-cost tool to assess carbapenemase-producing and non-producing in Gram-negative bacilli

Introduction: Carbapenemase-producing Gram-negative bacilli is a worldwide problem, which represent a health concern, because most of its resistance mechanisms are encoded by plasmids, therefore easily transmissible in hospital settings. Many methods have been proposed to detect such resistance, but screening is still challenging, recently the modified carbapenem inactivation method has shown promising results, however more studies need to be performed. Hence, this study aimed to evaluate the performance of the mCIM in carbapenem-resistant Enterobacteriaceae and nonfermenting Gram-negative bacilli isolates.
 Materials and methods: From a microbial collection with molecular characterization of carbapenemase genes previously conducted, 100 Gram-negative bacilli isolates were selected, fifty-two non-carbapenemase producing and 48 carbapenemase-producing isolates. The mCIM was performed according to the CLSI guidelines, and to assess the validity of the method sensitivity and specificity were calculated.
 Results: The sensitivity of the mCIM observed in this study was 96% (46/48) and the specificity was 96,2% (50/52). Most of the Gram-negative bacilli carrying a carbapenemase gene were mCIM-positive, moreover, in carbapenem-resistant isolates that do not produce a carbapenemase (Enterobacteriaceae and non-fermenters) the results of the mCIM were negatives.
 Conclusion: Overall the mCIM provides a low-cost alternative for the screening of carbapenemase-producing Gram-negative bacilli. Our findings highlight that mCIM is a sensitive and specific method to assess carbapenemase-producing in Gram negative bacilli non-fermenters and Enterobacteriaceae as Enterobacter cloacae.

Performance of BioFire Blood Culture Identification 2 Panel (BCID2) for the detection of bloodstream pathogens and their associated resistance markers: a systematic review and meta-analysis of diagnostic test accuracy studies

BackgroundEarly identification of bloodstream pathogens and their associated antimicrobial resistance may shorten time to optimal therapy in patients with sepsis. The BioFire Blood Culture Identification 2 Panel (BCID2) is a novel multiplex PCR detecting 43 targets directly from positive blood cultures, reducing turnaround times.MethodsWe have performed a systematic review and meta-analysis of diagnostic test accuracy studies to assess the BCID2 performance for pathogen identification and resistance markers detection compared to gold standard culture-based methods (including phenotypic and/or genotypic characterization).ResultsNine studies were identified reporting data to build 2 × 2 tables for each BCID2 target, including 2005 blood cultures. The pooled specificity of the assay was excellent (> 97%) across most subgroups of targets investigated, with a slightly broader confidence interval for S. epidermidis (98.1%, 95% CI 93.1 to 99.5). Pooled sensitivity was also high for the major determinants of bloodstream infection, including Enterobacterales (98.2%, 95% CI 96.3 to 99.1), S. aureus (96.0%, 95% CI 90.4 to 98.4), Streptococcus spp. (96.7%, 95% CI 92.8 to 98.5), P. aeruginosa (92.7%, 95% CI 83.1 to 97.0), E. faecalis (92.3%, 95% CI 83.5 to 96.6), as well as blaCTX-M (94.9, 95% CI 85.7 to 98.3), carbapenemases (94.9%, 95% CI 83.4 to 98.6) and mecA/C & MREJ (93.9%, 95% CI 83.0 to 98.0). Sensitivity for less common targets was slightly lower, possibly due to their under-representation in the included studies.ConclusionsBCID2 showed good performance for detecting major determinants of bloodstream infection and could support early antimicrobial treatment, especially for ESBL or carbapenemase-producing Gram-negative bacilli and methicillin-resistant S. aureus.

Insights on the performance of phenotypic tests versus genotypic tests for the detection of carbapenemase-producing Gram-negative bacilli in resource-limited settings

Background:Carbapenemase-producing Gram-negative (CPGN) bacteria impose life-threatening infections with limited treatment options. Rigor and rapid detection of CPGN-associated infections is usually associated with proper treatment and better disease prognosis. Accordingly, this study aimed at evaluating the phenotypic methods versus genotypic methods used for the detection of such pathogens and determining their sensitivity/specificity values.Methods:A total of 71 CPGN bacilli (30 Enterobacterales and 41 non-glucose-fermenting bacilli) were tested for the carbapenemase production by the major phenotypic approaches including, the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), combined disk test by EDTA (CDT) and blue-carba test (BCT). The obtained results were statistically analyzed and correlated to the obtained resistant genotypes that were determined by using polymerase chain reactions (PCR) for the detection of the major carbapenemase-encoding genes covering the three classes (Class A, B, and D) of carbapenemases.Results:In comparison to PCR, the overall sensitivity/specificity values for detection of carbapenemase-producing organism were 65.62%/100% for MHT, 68.65%/100% for mCIM, 55.22%/100% for CDT and 89.55%/75% for BCT. The sensitivity/specificity values for carbapenemase-producing Enterobacterales were, 74%100% for MHT, 51.72%/ 100% for mCIM, 62.07%/100% for CDT and 82.75%/100% for BCT. The sensitivity/specificity values for carbapenemase-producing non-glucose fermenting bacilli were, 62.16%/100% for MHT, 81.57%/100% for mCIM, 50/100% for CDT and 94.74%/66.66% for BCT. Considering these findings, BCT possess a relatively high performance for the efficient and rapid detection of carbapenemase producing isolates. Statistical analysis showed significant association (p < 0.05) between blaNDM and/or blaVIM genotypes with MHT/CDT; blaKPC/blaGIM genotypes with CDT and blaGIM genotype with BCT.Conclusion:The current study provides an update on the performance of the phenotypic tests which are varied depending on the tested bacterial genera and the type of the carbapenemase. The overall sensitivity/specificity values for detection of CPO were 65.62%/100% for MHT, 68.65%/100% for mCIM, 55.22%/100% for CDT and 89.55%/75% for BCT. Based on its respective diagnostic efficiency and rapid turnaround time, BCT is more likely to be recommended in a resource-limited settings particularly, when molecular tests are not available.

Synergistic antibacterial activity of silver nanoparticles biosynthesized by carbapenem-resistant Gram-negative bacilli

Nanotechnology is being investigated for its potential to improve nanomedicine for human health. The purpose of this study was to isolate carbapenemase-producing Gram-negative bacilli (CPGB), investigate the presence of carbapenemase resistance genes, determine their antibiogram and ability to biosynthesise silver nanoparticles (Ag NPs), and estimate the antibacterial activity of Acinetobacter baumannii-biosynthesised Ag NPs on CPGB alone and in combination with antibiotics. A total of 51 CPGBs were isolated from various specimens in the study. The automated Vitek-2 system was used to identify and test these strains' antimicrobial susceptibilities. The carbapenemase resistance genes were identified using a polymerase chain reaction (PCR). Under the CPGB, A. baumannii could biosynthesise Ag NPs. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), and field emission scanning electron were used to characterise Ag NPs. The antibacterial activity of Ag NP alone and in combination with antibiotics against CPGB was determined using the broth microdilution method, and their synergistic effect was determined using the checkerboard assay. blaNDM and blaOXA-48 were the most commonly reported, and 90% of the isolates produced multiple carbapenemase genes. Tigecycline proved to be the most effective anti-CPGB antibiotic. Isolates with more resistance genes were more resistant to antibiotics, and isolates with three genes (42%) had the most extensively drug-resistant patterns (38%). A significant relationship was discovered between genetic and antibiotic resistance patterns. Only A. baumannii produced Ag NPs out of all the isolates tested. Ag NPs with a size of 10 nm were confirmed by UV–visible spectroscopy, FT-IR, XRD, and TEM analysis. The Ag NPs were effective against CPGB, with minimum inhibitory concentrations ranging from 64 to 8 μg/ml on average. Surprisingly, the combination of Ag NPs and antibiotics demonstrated synergistic and partial synergistic activity (fractional inhibitory concentration between 0.13 and 0.56) against CPGB, as well as a significant reduction in antibiotic concentrations, particularly in the case of A. baumanii versus ceftriaxone (1024 to 4 μg/ml). The notable synergistic activity of Ag NPs with antibiotics represents a valuable nanomedicine that may find clinical application in the future as a combined remedy.

Faecal carriage of multidrug-resistant bacteria and associated risk factors: results from a point prevalence study.

Since 2003, incidences of carbapenemase-producing Gram-negative bacilli (CP-GNB) and vancomycin-resistant Enterococcus faecium (VRE) have steadily increased in France. We therefore conducted a point prevalence study to estimate carriage rates of CP-GNB, VRE and ESBL-producing Enterobacterales (ESBL-PE) and associated risk factors. Between September 2019 and January 2020, all inpatients hospitalized on a given day in 11 teaching hospitals in the Paris urban area were eligible. Patient interviews and rectal swab screening results were recorded by dedicated nurses. The swabs were plated onto selective chromogenic media and processed using the GeneXpert® system. Of 2396 patients, 364 (15.2%) yielded at least one multiresistant bacterial isolate, including 29 CP-GNB carriers (1.2%), 13 VRE carriers (0.5%) and 338 ESBL-PE carriers (14%). In 15 patients (4.4% of ESBL-PE carriers and 36.6% of CP-GNB/VRE carriers), concomitant CP-GNB/VRE and ESBL-PE carriage was observed. In 7/29 CP-GNB and 7/13 VRE carriers, carbapenemase production and vanA in the screening samples was only detected with Xpert® tests. The OXA-48 gene was predominant in 13/34 CP-GNB isolates from 29 carriers. From the 338 ESBL-PE carriers, 372 isolates were recovered, mainly Escherichia coli (61.2%). Among 379 children, 1.1% carried a CP-GNB/VRE strain, and 12.4% carried an ESBL strain. Previous hospitalization outside mainland France, previous antimicrobial treatment and previous ESBL-PE carriage were the main risk factors associated with CP-GNB and/or VRE carriage. The low CP-GNB and VRE prevalence likely reflects the French policy to limit intrahospital spread of CP-GNB and VRE strains.

Multi-Drug Resistance Profile, Prevalence of Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Gram Negative Bacilli Among Admitted Patients After Surgery with Suspected of Surgical Site Nosocomial Infection North East Ethiopia.

BackgroundAntibiotic resistance is becoming a global issue, with estimated 2.8 million people in the United States developing antibiotic-resistant diseases each year. The carriage of ESBL and Carbapenemase-producing Enterobacteriaceae among hospitalized patients is a threat to the future of antibiotic treatment.ObjectiveMulti-drug resistance profile, prevalence of extended-spectrum beta-lactamase and Carbapenemase-producing Gram-negative bacilli among admitted patients after surgery with suspected surgical site nosocomial infection north east Ethiopia.Material and MethodsA hospital-based cross-sectional study was conducted from April 2021 to February 2022. Socio-demographic and clinical data were assessed using a structured questionnaire. A total of 384 relevant clinical samples (pus, pus aspirates, and wound swabs) were collected aseptically and processed within 30 min by placing the swabs into the sterile test tubes having 0.5 mL of sterile normal saline. The samples were cultured on MacConkey agar, chocolate agar and blood agar, and species identification was done using standard biochemical tests. Disk diffusion antimicrobial sensitivity test was done on Mueller–Hinton agar. All the cefoxitin resistant Enterobacteriaceae isolates were checked for the presence of AmpC beta-lactamase using four cartridges of disk diffusion tablets. ESBL output validation was conducted by the combination disk test. The production of Carbapenemase was checked using modified carbapenem inactivation method.ResultsThe prevalence of significant bacterial nosocomial infection among surgical site infection 343 (89.32%). S. aureus 125 (36.4%) was predominant followed by E. coli 80 (23.3%) P. aeruginosa 31 (9.03%). The overall MDR rate of isolated bacteria was 251 (73.3%). About 150 (73.9%) bacteria were suspected for ESBL production and 67 (33%) AmpC beta-lactamase and 27 (13.3%) Carbapenemase-producing Gram-negative bacterial, respectively.Conclusions and RecommendationsThe severity of ESBL-PE was critical, and the CPE was alarming. Meropenem and imipenem were the most effective antibiotics against ESBL-producing Enterobacteriaceae. Therefore, strict infection prevention and control measures are needed.

Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Enterobacterales (CPE) pose one of the most serious antimicrobial resistance threats to public health worldwide. The outcome of CPE infection differs depending on the resistance mechanism. Therefore, rapid detection of CPE infection is essential for optimizing patient management. The carbapenem inactivation method (CIM) and modified CIM (mCIM) are standard methods for detecting CPE, but they usually require 24 h to generate results. Recently, an immunochromatographic assay, NG-Test CARBA 5, has become commercially available. It detects the five most common carbapenemase producers (KPC, IMP, NDM, VIM, and OXA-48) rapidly and accurately. We aimed to evaluate the diagnostic accuracy of NG-Test CARBA 5 for detecting carbapenemase-producing Gram-negative bacilli (CPGNB). We used 116 carbapenemase-producing strains and 48 non-carbapenemase-producing strains. Of the 116 carbapenemase-producing strains, 107 harboured genes for at least one of the five most common carbapenemases, KPC, IMP, NDM, VIM, and OXA-48-like. Forty-eight non-carbapenemase-producing strains, including carbapenem-resistant Enterobacterales, harboured genes for extended-spectrum β-lactamases (CTX-M groups [n=25] and SHV groups [n=2]) or plasmid-mediated AmpC β-lactamases (DHA [n=3], CMY-2 [n=2], and CFE-1 [n=1]). Antimicrobial susceptibility was tested using the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines. Of the 116 carbapenemase-producing strains, 79 were resistant to at least meropenem or imipenem. The sensitivity and specificity of the NG-Test CARBA 5 for the strains were 99.1 % (106 strains positive for 107 strains of the five most common carbapenemase producers) and 100 % (60 strains negative for other types of CPGNB [n=10] and non-CPGNB strains [n=48]), respectively. The carbapenemase-producing strain with a false-negative result produced IMP-66. The NG-Test CARBA 5 had high sensitivity and specificity for detecting carbapenemase-producing strains.

Phenotypic characterization of carbapenem non-susceptible gram-negative bacilli isolated from clinical specimens.

BackgroundMultidrug resistant, extremely drug-resistant, pan-drug resistant, carbapenem-resistant, and carbapenemase-producing gram-negative bacteria are becoming more common in health care settings and are posing a growing threat to public health.ObjectiveThe study was aimed to detect and phenotypically characterize carbapenem no- susceptible gram-negative bacilli at the Ethiopian Public Health Institute.Materials and methodsA prospective cross-sectional study was conducted from June 30, 2019, to May 30, 2020, at the national reference laboratory of the Ethiopian Public Health Institute. Clinical samples were collected, inoculated, and incubated for each sample in accordance with standard protocol. Antimicrobial susceptibility testing was conducted using Kirby-Bauer disk diffusion method. Identification was done using the traditional biochemical method. Multidrug-resistant and extensively drug-resistant isolates were classified using a standardized definition established by the European Centre for Disease Prevention and Control and the United States Centers for Disease Prevention and Control. Gram-negative organisms with reduced susceptibility to carbapenem antibiotics were considered candidate carbapenemase producers and subjected to modified carbapenem inactivation and simplified carbapenem inactivation methods. Meropenem with EDTA was used to differentiate metallo-β-lactamase (MBL) from serine carbapenemase. Meropenem (MRP)/meropenem + phenylboronic acid (MBO) were used to differentiate Klebsiella pneumoniae carbapenemase (KPC) from other serine carbapenemase producing gram-negative organisms.ResultsA total of 1,337 clinical specimens were analyzed, of which 429 gram-negative bacterial isolates were recovered. Out of 429 isolates, 319, 74, and 36 were Enterobacterales, Acinetobacter species, and Pseudomonas aeruginosa respectively. In our study, the prevalence of multidrug-resistant, extensively drug-resistant, carbapenemase-producing, and carbapenem nonsusceptible gram-negative bacilli were 45.2%, 7.7%, 5.4%, and 15.4% respectively. Out of 429 isolates, 66 demonstrated reduced susceptibility to the antibiotics meropenem and imipenem. These isolates were tested for carbapenemase production of which 34.8% (23/66) were carbapenemase producers. Out of 23 carbapenemase positive gram-negative bacteria, ten (10) and thirteen (13) were metallo-beta-lactamase and serine carbapenemase respectively. Three of 13 serine carbapenemase positive organisms were Klebsiella pneumoniae carbapenemase.ConclusionThis study revealed an alarming level of antimicrobial resistance (AMR), with a high prevalence of multidrug-resistant (MDR) and extremely drug-resistant, carbapenemase-producing gram-negative bacteria, particularly among intensive care unit patients at the health facility level. These findings point to a scenario in which clinical management of infected patients becomes increasingly difficult and necessitates the use of “last-resort” antimicrobials likely exacerbating the magnitude of the global AMR crisis. This mandates robust AMR monitoring and an infection prevention and control program.

Evaluación de métodos fenotípicos para la detección de carbapenemasas aplicables a laboratorios de poca complejidad

The spread of carbapenemase-producing gram-negative bacilli is a global public health problem. Several authors have proposed phenotypic assays to presumptively detect these enzymes applicable to low and medium complexity laboratories. In the present study, we have developed and compared different phenotypic techniques using strains genetically identified as carbapenemase-producing. All the tested methods detected the presence of carbapenemases. The carbapenem inactivation method (MIC) and the modified carbapenem inactivation method with and without EDTA (mMIC-eMIC) were the simplest and easiest to interpret but their disadvantage was on the time required to obtain results. The direct Carba NP and Carba-Blue colourimetric methods were the fastest but they depend on reagent preparation and accurate pH adjustment of the solutions. Synergy methods with EDTA discs, boronic acid and the Triton Hodge Test (THT) require technical expertise to evaluate true synergism. Whereas, the Disk Carbapenemase Test (DCT) was the method that presented the greatest technical difficulties.

Prevalence and characterisation of carbapenemase encoding genes in multidrug-resistant Gram-negative bacilli.

Emerging worldwide in the past decade, there has been a significant increase in multidrug-resistant bacteria from serious nosocomial infections, especially carbapenemase-producing Gram-negative bacilli that have emerged worldwide. The objective of this study is to investigate carbapenem resistance in Gram-negative bacilli bacteria using phenotypic detection, antimicrobial resistance profiles and genotypic characterisation methods. 200 Gram-negative bacilli isolates were collected from different clinical specimens. All clinical samples were exposed to isolation and identification of significant pathogens applying bacteriological examination and an automated Vitek-2 system. The isolates were subjected to susceptibility tests by the Vitek-2 automated system and those isolates that were resistant to beta-lactam drugs, including carbapenems, third-generation cephalosporines or cefoxitin, were selected for phenotyping using Carba plus disc system assay for detection of carbapenemase-producing isolates. These isolates were further confirmed by molecular detection. PCR was used for the detection carbapenem-resistant genes (OXA-48, IMP, NDM, VIM, and KPC). 110 (55%) of 200 Gram-negative bacilli were identified as beta-lactam-resistant isolates. The frequency of carbapenem-resistant isolates was calculated to be 30.9% (n = 34/110). A collection totalling 65/110 (59%) isolates were identified as carbapenemase producers by phenotypic method. Moreover, among the 65 carbapenemase-producing Gram-negative isolates with a positive phenotype-based result, 30 (46%), 20 (30%) and 18 (27%) isolates were positive for OXA-48, KPC and MBL enzymes, respectively, as well as the production of 27% of AmpC with porin loss. Tigecycline was the most effective antibiotic that affected 70% of MDR isolates, but high rates of resistance were detected to other tested antimicrobials. Of interest, a high incidence of MDR, XDR and PDR profiles were observed among all carbapenemase-producing isolates. 36% (24/65) of the tested isolates were MDR to 3 to 5 antimicrobial classes. 29% (17/65) of the recovered isolates were XDR to 6 to 7 antimicrobial classes. Alarmingly, 24% (16/65) of isolates displayed PDR to all the tested 8 antimicrobial classes. Genotype assay, including 53 phenotypically confirmed carbapenemase-producing isolates of Gram-negative bacilli, found 51(96%) isolates were harbouring one or more genes. The most common carbapenemase gene was bla NDM 83% (44/53) followed by bla OXA-48 75% (40/53), bla VIM 49% (26/53) and bla IMP 43% (23/53), while the gene bla KPC was least frequent 7% (4/53). 92% (46/51) of isolates were involved in the production of more than one carbapenemase gene. This study demonstrated the emergence of carbapenemase-producing Gram-negative pathogens implicated in healthcare-related infections. Accurate identification of carbapenem-resistant bacterial pathogens is essential for patient treatment, as well as the development of appropriate contamination control measures to limit the rapid spread of pathogens. Tigecycline exhibited potent antimicrobial activity against MDR, XDR and PDR-producing strains that establish a threatening alert which indicates the complex therapy of infections caused by these pathogens.

Investigation of Extended-Spectrum β-Lactamase and Carbapenemase Producing Gram-Negative Bacilli in Rectal Swabs Collected from Neonates and Their Associated Factors in Neonatal Intensive Care Units of Southern Ethiopia.

BackgroundCurrently extended-spectrum β-lactamase (ESβL) and carbapenemase producing gram-negative bacteria are the greatest concern among the neonatal population with very limited therapeutic options. The aim of this study was to assess the prevalence of ESβL and carbapenemase producing gram-negative bacilli, associated factors and antimicrobial resistance patterns among neonates in intensive care units.MethodsAn institutional-based cross-sectional study was conducted from February to June 2021 on 212 neonates in intensive care units. Risk factors data were collected by using a well-designed questionnaire. A rectal swab sample was collected using a sterile cotton swab and inoculated on MacConkey agar. Bacterial isolates were identified using various biochemical tests. ESβL and carbapenemase were first screened by indicator cephalosporins (cefotaxime (30µg) and ceftazidine (30µg)) and carbapenem (meropenem and ertapenem), respectively. ESβL and carbapenemase were confirmed by a double-disk synergy test and modified carbapenem inactivation methods, respectively. SPSS version 21.0 was used for data analysis. A P-value ≤ 0.05 was considered as statistically significant.ResultsThe overall prevalence of ESβL-producing gram-negative bacilli was 72/212 (34%). The predominant ESβL-producing isolate was Klebsiella pneumoniae 23/72 (31.9%) followed by Escherichia coli 17/72 (23.6%). Five (2.4%) carbapenemase-producing gram-negative bacilli were isolated. ESβL-producing isolates showed a high resistance against ampicillin 72/72 (100%), augmentin 69/72 (95.8%) and gentamycin 57/72 (79.2%). The majority 63/72 (87.5%) of isolated ESβL-producing gram-negative bacilli were multi-drug resistant (MDR). Rectal carriage of ESβL by neonates showed a statistically significant association with endotracheal intubation (p = 0.001; AOR = 4.2; 96% CI = (1.8–9.5)), treatment with ampicillin+gentamycin (p = 0.004; AOR = 3.3; 95% CI = (1.5–7.6)) and staying in a neonatal intensive care unit (NICU) between 11 and 20 days (p = 0.042; AOR = 2; 95% CI = (1.0–4.5)).ConclusionA high prevalence of ESβL-producing bacterial isolates was observed for commonly used antibiotics which needs further attention. Therefore, continuous and regular follow-ups of drug resistance patterns is important for the proper treatment and management of ESβL and carbapenemase producing gram-negative bacilli.

Comparison of the performance of three carbapenem inactivation methods for the detection of carbapenemase-producing gram-negative bacilli

IntroductionThe carbapenem inactivation method test (CIM) was developed as a method for detecting carbapenemase-producing Gram-negative bacilli, and the modified CIM (mCIM) was recommended by the CLSI for as an improved method in M100-S27. However, few studies have evaluated the influence of bacterial species and genotype on its sensitivity and specificity. In this study, we evaluate the performance of these improved modified CIM methods with mCIM. MethodsAs strains, clinical isolates from Naga Municipal Hospital and stored strains from the Study of Bacterial Resistance in the Kinki Region of Japan were used. The mCIM, CIM-Tris, and simple CIM (sCIM) test methods were applied to 120 Enterobacterales, 40 Pseudomonas aeruginosa, and 37 Acinetobacter spp. The procedure and criteria for each method were based on the original papers and the CLSI M − 100 S27 documents. ResultsThe sensitivity of the test methods in the detection of carbapenemase in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. was as follows: mCIM, 98.9%, 90.0%, and 76.5%, respectively; CIM-Tris, 94.4%, 100%, 100%; and sCIM 98.9%, 85.0%, 76.5%. All methods showed 100% specificity in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. Each method performed well in the detection of metallo β-lactamase-producing strains, however, the sensitivity tended to be low in the detection of the organisms producing serine-type carbapenemase, such as GES, OXA-23, and OXA-51. ConclusionsCare must be taken when selecting test methods because the sensitivity of the detection differs depending on the bacterial species and genotype.

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1

PurposeCarbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India. MethodsFifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis. ResultsIsolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 ​% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 ​h detected 92.3 ​% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 ​h at cut off 17 ​mm while for NDM-1 CIM was the test of choice at 16 ​mm. ConclusionCIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 ​h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 ​mm was considered adequate for detection of OXA-48-like and 16 ​mm of NDM-1 by mCIM at 4 ​h and CIM respectively.

Four-Hour Immunochromatographic Detection of Intestinal Carriage of Carbapenemase-Producing Enterobacteriaceae: a Validation Study.

The increasing incidence of carbapenemase-producing Gram-negative bacilli (C-PGNB) represents a major public health challenge. Rapid detection of digestive colonization with C-PGNB is fundamental to control their spread. We performed the validation of a rapid protocol for C-PGNB detection directly on rectal swabs. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 O.K.N.V. K-SeT test on the bacterial pellet obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on a calibrated sample suspension and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization (n = 48) and controls (patients with extended-spectrum beta-lactamase [ESBL] colonization [n = 48] and without carbapenemase/ESBL [n = 48]). The protocol detected, with 100% sensitivity, the presence of the 15 OXA-48-, 14 KPC-, 13 NDM-, and 10 VIM-producing GNB from 103 CFU/ml. The limit of detection was 2 × 102 CFU/ml. Among the 48 C-PGNB-containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumannii strain and 1 OXA-48-producing Escherichia coli strain. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% (95% confidence interval [CI], 87.7 to 100) and 100% (95% CI, 96.2 to 100). The negative likelihood ratio was 0.04 (95% CI, 0.01 to 0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with carbapenemase-producing Enterobacteriaceae in 4 h without any requirement for specific equipment.

Detection and characterization of carbapenem resistant Gram\u2010negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan

BackgroundAntimicrobial resistance (AMR) poses a complex threat to global health security and universal health coverage. Recently, nosocomial infections with carbapenemase-producing Gram-negative bacilli (GNB) is increasing worldwide. We report the molecular characterization and detection of genes associated with carbapenemase producing Gram negative bacteria isolated from hospitalized patients at Soba University Hospital (SUH) in Khartoum State, Sudan.ResultsBetween October 2016 and February 2017, a total of 206 GNB clinical specimens were collected from hospitalized patients in SUH. Of 206 carbapenem resistance isolates, 171 (83 %) were confirmed as phenotypically resistant and 121 (58.7 %) isolates harboured one or more carbapenemase genes. New Delhi metallo-β-lactamase (NDM) types were the most predominant genes, blaNDM 107(52 %), followed by blaIMP 7 (3.4 %), blaOXA-48 5(2.4 %) and blaVIM 2 (0.9 %). Co-resistance genes with NDM producing GNB were detected in 87 (81.3 %) of all blaNDM producing isolates. NDM-1 was the most frequent subtype observed in 75 (70 %) blaNDM producing isolates. The highest percentage of resistance was recorded in ampicillin (98 %), cephalexin (93.5 %) amoxicillin clavulanic acid (90 %), cefotaxime (89.7 %), ceftriaxone (88.4 %), ceftazidime (84.2 %), sulfamethoxazole-trimethoprim (78.4 %) and nitrofurantoin (75.2 %), aztreonam (66 %) and temocillin (64 %). A close correlation between phenotypic and carbapenemase genes detection in all GNB was observed.ConclusionsThe frequency of carbapenemase producing bacilli was found to be high in SUH. NDM was found to be the most prevalent carbapenemase gene among clinical isolates. Close surveillance across all hospitals in Sudan is required. The relative distribution of carbapenemase genes among GNB in nosocomial infections in Africa needs to be defined.