Detection Of Carbapenemase Genes Research Articles

P-1119. The role of Carbapenemase gene detection and synergy testing in invasive infections caused by Carbapenem resistant Enterobacterales in the pediatric population at a quaternary hospital in South India

Abstract Background The incidence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) infections in children is increasing globally. However, there are limited studies that address clinical, microbiological profiles, molecular and synergy testing of these CRE infections, in this population. Our study was aimed at studying the clinical profile, microbiological and molecular diagnoses, and synergy testing of CRE bacteremia in children admitted to the intensive care unit (ICU). Methods Medical records of children admitted to the ICU with CRE bacteraemia over a year were reviewed. Their clinical background, microbiological diagnoses, molecular profile, antibiotic resistance patterns, synergy testing, and clinical outcomes were analyzed. Carbapenemase genes were detected using Gene X pert (Cepheid) and synergy testing was carried out on those isolates with New Delhi Metalloproteinase (NDM). NDM-1 or NDM + Oxacillinase (OXA) OXA-48 genes. The results were expressed as fractional inhibitory concentration index (FICI) and the same was correlated with treatment using Ceftazidime- Avibactam+/- Aztreonam. Results 98 children with blood stream infections (40 in neonatal ICU and 58 pediatric ICU) were identified. K. pneumoniae accounted for a majority of BSI’s (n-34 in neonatal ICU and n- 47 in pediatric ICU) with most of them expressing the NDM gene (50%). The overall Colistin resistance across both ICU’s was (MIC > 2 ug ml) was 0.03%. Synergy to Ceftazidime-Avibactam- Aztreonam could be demonstrated in a majority of CREs which were NDM / NDM+OXA-48 producers (82.3% K. pneumoniae in neonatal ICU and 90% in pediatric ICU). Eight neonates (20%) and seventeen children (29%) succumbed due to other comorbidities, all of whom had NDM Klebsiella pneumoniae bacteraemia (n-17, 100%). Conclusion NDM producing Klebsiella pneumoniae remains the commonest identified BSI in neonatal and paediatric patients in the intensive care and was isolated in all children with adverse outcomes. Infection control, antimicrobial stewardship protocols and Carbapenemase gene detection with targeted synergy and directed therapy is recommended for favorable clinical outcomes of BSI’s in the critically ill children. Disclosures All Authors: No reported disclosures

P-1354. Carbapenemase Gene Detection in Clinically Significant Pseudomonas aeruginosa Isolates from A Tertiary Care Health Center: A Prospective Study

Abstract Background Pseudomonas aeruginosa is a leading pathogen causing widespread hospital acquired infections worldwide. As WHO has also designated Carbapenem resistant Pseudomonas aeruginosa as one of three Critical Priority Pathogen, we decided to conduct a study on the Pseudomonas aeruginosa isolates to find out the presence of carbapenemase genes using RT-PCR and to find out the relation between the phenotypic assessment of carbapenem resistance with genotypic detection of carbapenemase genes. Methods Total 41 patients were included in this prospective observational study done for 6 months from 1st July 2023 to 31st December 2023. Pseudomonas aeruginosa isolates from the patients with age >18 years, ICU admission for more than 48 hours and absence of any coinfection were included and those patients with polymicrobial infections and multisite infections were excluded. Isolates were obtained from the blood, respiratory and pyogenic samples and antimicrobial susceptibility was done by VITEK for various antimicrobials and broth microdilution for colistin. The bacterial colony was used for DNA extraction using Bacterial Genomic DNA Extraction Kit in accordance with the manufacturer’s instruction. Multiplex RT PCR was done to detect genes blaVIM, blaNDM, blaKPC and blaIMP. Results Out of 41 isolates, as depicted in figure 1, blaIMP was most common gene found in 26(63.41%), followed by blaNDM in 20(48.7%) isolates. Combination of blaNDM and blaIMP was found in 12 (29.2%) and blaNDM with blaKPC in 3(7.3%) patients. As shown in table 1 even in isolates which were found pan sensitive to all antimicrobials in VITEK were also harboring genes for carbapenemase. blaIMP was found in 9(69%) of these isolates out of 13 pan sensitive ones. Attributable mortality according to phenotypic resistance is described in table 1. Figure 2 is amplification plot of blaNDM by RT PCR of one of the isolates. Conclusion We found presence of blaNDM and blaIMP genes in our isolates of Pseudomonas aeruginosa as compared to global presence of blaKPC and blaVIM. High prevalence of carbapenemase genes even in pan-sensitive isolates can be attributed to excessive use of carbapenems and larger studies are required to find out its impact on clinical outcome. Disclosures All Authors: No reported disclosures

Prevalence and Molecular Characterization of Carbapenemase-Producing Multidrug-Resistant Bacteria in Diabetic Foot Ulcer Infections.

Background: Diabetic foot ulcers (DFUs) represent severe complications in diabetic patients, often leading to chronic infections and potentially resulting in nontraumatic lower-limb amputations. The increasing incidence of multidrug-resistant (MDR) bacteria in DFUs complicates treatment strategies and worsens patient prognosis. Among these pathogens, carbapenemase-producing pathogens have emerged as particularly concerning owing to their resistance to β-lactam antibiotics, including carbapenems. Methods: This study evaluated the prevalence of MDR bacteria, specifically carbapenemase-producing pathogens, in DFU infections. A total of 200 clinical isolates from DFU patients were analyzed via phenotypic assays, including the modified Hodge test (MHT) and the Carba NP test, alongside molecular techniques to detect carbapenemase-encoding genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48). Results: Among the isolates, 51.7% were confirmed to be carbapenemase producers. The key identified pathogens included Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. The most commonly detected carbapenemase genes were blaKPC (27.6%) and blaNDM (24.1%). Carbapenemase-producing isolates presented high resistance to β-lactam antibiotics, whereas non-carbapenemase-producing isolates presented resistance through mechanisms such as porin loss and efflux pumps. Conclusions: The findings of this study highlight the significant burden of MDR infections, particularly carbapenemase-producing organisms, in DFUs. MDR infections were strongly associated with critical clinical parameters, including pyrexia (p = 0.017), recent antibiotic use (p = 0.003), and the severity of infections. Notably, the need for minor amputations was much higher in MDR cases (p < 0.001), as was the need for major amputations (p < 0.001). MDR infections were also strongly associated with polymicrobial infections (p < 0.001). Furthermore, Wagner ulcer grade ≥II was more common in MDR cases (p = 0.002). These results emphasize the urgent need for enhanced microbiological surveillance and the development of tailored antimicrobial strategies to combat MDR pathogens effectively. Given the high prevalence of carbapenem resistance, there is an immediate need to explore novel therapeutic options to improve clinical outcomes for diabetic patients with DFUs.

Epidemiological and genetic characteristics of clinical carbapenemase-producing Enterobacterales isolates from Batna hospitals in Algeria

BackgroundCarbapenemase-producing Enterobacterales isolates are associated with significant mortality and have emerged as a major problem in healthcare settings worldwide.ObjectiveOur aim was to investigate the epidemiological and genotypic characteristics of carbapenemase-positive Enterobacterales isolates from patients hospitalised in three hospitals in the city of Batna, Algeria.MethodsBetween 2016 and 2019, a total of 5,316 clinical isolates were obtained. The collected isolates were identified using the VITEK-2 system. Demographic and microbiological data were collected as well as the antimicrobial susceptibility testing, phenotypic and molecular characterisation of carbapenemase and mcr-1 genes were performed.ResultsOut of the 5,316 isolates, 201 were carbapenem-resistant Enterobacterales isolates and 179 of them (89.05%) were positive for the production of carbapenemase, of which Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae were the most common. The blaOXA−48−like gene alone was detected in 147 isolates (82.12%) moreover, the blaNDM gene was detected in ten isolates (5.59%). Dual and triple combinations of carbapenemase genes were also observed here for the first time in Algeria: blaVIM and blaOXA−48−like; blaKPC, blaVIM and blaOXA−48−like; blaVIM and blaNDM; blaKPC, blaNDM and blaVIM; blaNDM and blaOXA−48−like genes. In addition, resistance to both colistin and carbapenem antibiotics was detected in eight isolates, however none of them was positive for the mcr-1 gene.ConclusionThis is the first study reporting the detection of carbapenemase genes in Klebsiella aerogenes, Enterobacter sakazakii, Raoultella ornithinolytica, Serratia ficaria, and Serratia marcescens and specific carbapenemase gene combinations in Algeria. The present study revealed that blaOXA−48−like were found to be the predominant carbapenemase genes in Batna hospitals.

Investigation of the synergistic effect ofceftazidime-avibactam and aztreonam combination on carbapenem-resistant Klebsiella pneumoniae isolates with 3 different methods.

Treatment options are limited for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates due to the production of metallo-β-lactamase (MBL). The ceftazidime-avibactam (CZA)/ aztreonam (ATM) combination represents a new therapeutic approach in MBL-positive isolates. Our study aims to determine distribution of carbapenemase genes in CRKP isolates and to investigate the in vitro synergistic effect of the CZA/ATM combination.Our study included 48 CRKP strains isolated from various clinical samples. Identification was performed using MALDI-TOF MS (bioMérieux, France), and susceptibility was tested with Vitek-2 (bioMérieux). The susceptibility to CZA and ATM was determined using CZA 30/20 µg and ATM 30 µg (Oxoid™,UK) disks. Carbapenemase genes VIM, NDM, IMP, KPC, OXA-23, OXA-58, OXA-48, and OXA-51 were investigated in only 44 isolates using the Bio-Speedy Carbapenem resistance qPCR (Bioexen, Turkiye) kit. Synergy testing was evaluated with double disk diffusion, gradient strip (bioMérieux)/disk diffusion, and broth disk elution methods.Out of 48 carbapenem-resistant isolates, 40 (83.3%) isolates showed resistance to CZA and 46 (95.8%) to aztreonam. Synergy was detected with all three methods in all isolates identified as resistant to CZA, CZA-sensitive isolates were not included in this evaluation. The most frequently detected carbapenemase genes were NDM+OXA-48, found in 28 (63.6%) of the isolates.Although the NDM+OXA-48 coexistence predominates in our center, in vitro synergy between CZA and ATM was detected in all of CZA-resistant isolates. Performing the CZA+ATM synergy test and reporting the result is crucial for choosing appropriate treatment in CRKP infection.

Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30-40 min for RCCS reaction (a 20 min RPA reaction with a 10-20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.

Emergence of Escherichia coli ST131 carrying carbapenemase genes, European Union/European Economic Area, August 2012 to May 2024.

Analysis of 594 isolates of Escherichia coli sequence type (ST)131 and its single locus variants carrying carbapenemase genes from 17 European Union/European Economic Area countries revealed acquisition of 18 carbapenemase variants, mainly in ST131 clades A and C. Most frequent were bla OXA-244 (n = 230) and bla OXA-48 (n = 224), detected in 14 and 12 countries, respectively. Isolates carrying bla OXA-244 have increased rapidly since 2021. The increasing detection of carbapenemase genes in the E. coli high-risk lineage ST131 is a public health concern.

Evaluation of the CARBA PAcE test, a colorimetric imipenem hydrolysis test for rapid detection of carbapenemase activity.

There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we evaluated a colorimetric imipenem hydrolysis test, called the CARBA PAcE test, to detect carbapenemase-producing Gram-negative bacteria (GNB). We tested a collection of 270 GNB isolates with a characterized carbapenemase content. Our testing set included 205 carbapenemase-producing, carbapenemase-resistant Enterobacterales (CP CRE) with 40 Klebsiella pneumoniae carbapenemase (KPC), 49 New Delhi metallo beta lactamase (NDM), 49 OXA-48-like, 15 Verona integron-mediated metallo-β-lactamase (VIM), three IMP, 43 IMI, six isolates producing more than one carbapenemase, and 65 non-carbapenemase-producing Enterobacterales (20 ESBL producers, 35 non-carbapenemase-producing, carbapenem-resistant [non-CP CRE], and 10 carbapenem-susceptible Enterobacterales [non-CP, non-CSE, third-generation cephalosporin and carbapenem susceptible]). We compared the performances of the CARBA PAcE test, the qualitative colorimetric β-CARBA test, and the modified CarbaNP test to a gold standard of carbapenemase gene detection by PCR. Specificities of all tests were high: 95.4% (62/65) for CARBA PAcE test, 98.5% (64/65) for β-CARBA test, and 100% (65/65) for the modified CarbaNP test. Sensitivity varied by carbapenemase: all three tests had a sensitivity of 100% for NDM, VIM, and IMP and 97.5% for KPC. Sensitivity to detect IMI was 0% for the CARBA PAcE and β-CARBA tests and 11.6% for the modified CarbaNP test. Sensitivity to detect OXA-48-like was 89.7% for the CARBA PAcE test, 87.7% for the β-CARBA test, and 14.2% for the modified CarbaNP test. Reading the results of the CARBA PAcE assay was difficult. The CARBA PAcE assay is highly sensitive for detecting NDM, VIM, IMP, and KPC, but slightly less sensitive for OXA-48-like. It does not detect IMI. It is highly specific, and its overall diagnostic accuracy is similar to that of β-CARBA. Its operational advantages are rapid turnaround time, ease of use, and long shelf life, but reading of results is subjective.IMPORTANCEWe evaluated the ability of the CARBA PAcE test to detect carbapenemases in 274 Gram-negative isolates with a known carbapenemase content. Specificity was high for all carbapenemases tested (96.9%). Sensitivity was high for KPC, NDM, VIM, and IMP (97.5-100%); but lower for OXA-48-like (89.7%). Activity of IMI could not be detected. Taken together, our results indicate that CARBA PAcE is a useful alternative in regions where NDM and KPC are predominant. The limitations of the test are difficulty in reading results and incompatibility with mSuperCARBA.

Carbapenemase-Producing Escherichia coli: Comparison of a Novel Rapid Lateral Flow Assay With the Polymerase Chain Reaction (PCR) and Antimicrobial Resistance Pattern.

Background In critically ill patients, carbapenems are often used as the last line of treatment. Carbapenem-resistant Enterobacterales (CRE) present an extreme challenge to treatment due to their resistance to various antibiotics. Optimal therapy for patients and infection control relies on the early and accurate diagnosis of these infections. The K.N.I.V.O. Detection K-Set is a newly developed immunological rapid test developed to identify the presence of carbapenemase in Gram-negative bacteria resistant tomultiple drugs. Objectives This study evaluates a new K.N.I.V.O. Detection K-Set and its application for the rapid detection of isolates of multidrug-resistant Escherichia coli (MDR E. coli) that produce carbapenemase. This test aims to compare the test's performance to the polymerase chain reaction (PCR) method. Methods The study included 150 MDR E. coli isolates that were confirmed to be resistant to at least three groups of antibiotics, including carbapenems. The test followed the manufacturer's instructions using the K.N.I.V.O. Detection K-Set. The outcomes were compared with carbapenemase gene detection (bla-KPC, bla-NDM, bla-OXA-48, bla -VIM, and bla -IMP) using the PCR. The K-Set's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated and studied. Results The K.N.I.V.O. Detection K-Set showed highly effective diagnostic performance with a 97.1% sensitivity, 97.5% specificity, 97.1% positive predictive value, and 98.7% negative predictive value. Seventy-eight of the 150 isolates were proven to be producers of carbapenemase, with 68 of those cases having an accurate identification. The remaining isolates were found to be non-producers. Within 15 minutes, the rapid testprovided results. Conclusion The K.N.I.V.O. Detection K-Set is an effective and rapid method for identifying carbapenemase producers among MDR E. coli isolates. Its rapid processing time, associated with its high sensitivity and specificity, indicatesthat it can increase the effectiveness of diagnostic laboratories and better patient care in clinical settings. Implementing such rapid screenings could be vital for controlling the spread of drug-resistant infections and enhancing antimicrobial stewardship. This also ensures that patients receive timely treatment and effective care.

Molecular Detection of Carbapenemase Genes among Carbapenem Resistant Gram-Negative Bacilli in Tertiary Care Hospital, Bangladesh

Molecular Detection of Carbapenemase Genes among Carbapenem Resistant Gram-Negative Bacilli in Tertiary Care Hospital, Bangladesh

Comparative In Vitro Activity of Ceftazidime-Avibactam, Imipenem-Relebactam, and Meropenem-Vaborbactam against Carbapenem-Resistant Clinical Isolates of Klebsiella pneumoniae and Pseudomonas aeruginosa.

Co-infection with carbapenem-resistant Klebsiella pneumoniae (CRKP) and Pseudomonas aeruginosa (CRPA) is associated with poor outcomes and historically relied on combination therapy with toxic agents for management. However, several novel β-lactam/β-lactamase inhibitor combination agents have been developed, offering potential monotherapy options. Here, we compare the in vitro activity of ceftazidime-avibactam (CZA), imipenem-relebactam (IRL), and meropenem-vaborbactam (MVB) against both CRKP and CRPA clinical isolates. Minimum inhibitory concentrations (MICs) for each agent were determined using broth microdilution. Carbapenemase gene detection was performed for representative isolates of varying carbapenem resistance phenotypes. IRL demonstrated excellent activity against CRKP and CRPA with susceptibility rates at 95.8% and 91.7%, respectively. While CZA and MVB showed comparable susceptibility to IRL against CRKP (93.8%), susceptibility of CRPA to CZA was modest at 79.2%, whereas most CRPA strains were resistant to MVB. Of the 35 CRKP isolates tested, 91.4% (32/35) carried a blaKPC gene. Only 1 of 37 (2.7%) CRPA isolates tested carried a blaVIM gene, which conferred phenotypic resistance to all three agents. None of the CRKP strains were cross-resistant to all three agents. Source of infection and co-infection did not significantly influence antimicrobial activity for IRL and CZA; none of the CRPA isolates from co-infected patients were susceptible to MVB. Our results suggest that novel β-lactam agents with antipseudomonal activity and stability against carbapenemases, such as IRL and CZA, offer potential monotherapy options for the treatment of co-infection involving both CRKP and CRPA, but not MVB.

Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

Occurrence of Carbapenem-Resistant Enterobacteriaceae (Cre) Abhorring Bla Imp, Bla Kpc And Bla Oxa-48 Genes Recovered From Ready -To- Dispose Poultry Wastes In Osogbo, Osun State

The uncontrolled use of antimicrobials in animal husbandry such as poultry for prophylaxis reasons is to increase output for monetary gains which have led to several acquired community infections with high mortality and morbidity rates. This study is however aimed at investigating the occurrence, prevalence, and molecular detection of carbapenemase genes of carbapenem-resistant Enterobacteriaceae (CRE) bacteria isolated from poultry liters in Osogbo, Osun State Nigeria. Twenty ready-to-pack poultry waste samples were plated for the isolation of Enterobacteriaceae on MacConkey and Brilliance Escherichia coli agar. Disc diffusion method was employed for screening carbapenem resistant isolates while resistant genes were detected using carbapenem resistant gene primers. Forty-six Enterobacteriaeceae were isolated namely: Kluyvera ascorbate (28.3%), Klebsiella oxytoca (10.9%), Citrobacter koseri (8.7%), Klebsiella aereogenes, Raoultell (Klebsiella) planticola, Serratia nematodipila (6.5%), Trabulsiella laguamensis, Salmonella spp. (diarizonae), Proteus vulgaris, Citrobacter farmeri (4.3%), of which Edwardsiella tarda, Providencia rustigianii, Citrobacter freundii, Enterobacter cloacae, Cedecea lapagei, Serratia liquefaciens and Dickeyachry santhemi was (2.2%) respectively. 52.2% of the isolates were carbapenem resistant while 92% of the CRE had bla IMP genes. Only one CRE (Trabulsiella guamensis) was positive to bla KPC gene while four 16.6% of the isolates namely: Citrobacter koseri, Raoultell (Klebsiella) planticola, Serratia nematodipila, Citrobacter freundiu and Citrobacter koseri were positive to blaOXA-48 gene. Therefore, this study infers the high prevalence of CRE isolates in poultry litters as a public health concern for the indiscrimate use of carbapenem.&#x0D; Stamford Journal of Microbiology, 2023. Vol. 13, Issue 1, p. 15-20

Detection of Carbapenemase Genes by Molecular Method among Gram-Negative Bacilli Isolates from Tertiary Care Hospital in Dhaka, Bangladesh

Background: Imipenem resistance in Gram-negative bacilli is a global epidemic that is increasing day by day.To warn this global epidemic, identification and ongoing surveillance of carbapenem-resistant genes among Gram-negative bacilli needed.Objectives: This cross-sectional study was performed todetect the imipenem resistant genes among Gram-negative bacilli isolated from different samples in Dhaka medical college hospital.Methods: About 300 samples (wound swab, urine, endotracheal aspirate, blood, and sputum) were collected from July 2015 to June 2016. Among them, 204 isolates were Gram negative bacilli. Eighty imipenem resistant Gram-negative bacilli were isolated by disc diffusion method. Among them, carbapenem resistant genes (blaNDM-1, blaKPC, VIM, IMP) were detected by PCR.Results: A total 300 samples were analyzed. Out of 204 Gram negative bacilli,80 (39.21%) imipenem resistance was detected by the Disc Diffusion method. Out of 80 imipenem resistant organisms, 42 (52.5%) were positive for blaNDM-1, 6 (7.34%) were positive for blaKPC, 29 (36.25%) were positive for VIM, 13 (16.25%) were positive for IMP.Conclusion: This study illustrates the emergence of carbapenemase genes producing Gram negative bacilli isolates from patients. Close surveillance across all hospitals in Bangladesh is required. J Shaheed Suhrawardy Med Coll 2022; 14(1): 3-7

Intense Intestinal Carriage of Carbapenemase-Producing Klebsiella pneumoniae Co-harboring OXA-48, KPC, VIM, and NDM Among Preterm Neonates in a Moroccan Neonatal Intensive Care Unit.

This study aimed to investigate the prevalence and the carbapenemase production ability of Klebsiella pneumoniae isolates from premature neonates' intestinal tracts in a Moroccan neonatal intensive care unit Methodology: Active rectal screening was performed among 339 preterm infants. The collected isolates were subjected to antibiotic susceptibility testing, phenotypic analysis of carbapenemase production, and molecular detection of carbapenemase genes. Out of 293 K. pneumoniae isolates collected, 31.05% (91) were resistant to carbapenem and produced carbapenemase, resulting in a 22.12% rate of intestinal carriage. Among the carbapenem-resistant K. pneumoniae isolates, 40.65% (37) had co-harbored carbapenemase genes. All isolates contained the blaOXA-48 gene, and the blaNDM, blaVIM, and blaKPC genes were detected in 30.76%, 9.89%, and 2.19% of the isolates, respectively. Out of 30.76% of these isolates had both the blaOXA-48 and blaNDM genes, 8.79% had both blaOXA-48 and blaVIM, and only 2.20% had both blaOXA-48 and blaKPC genes. Furthermore, 88.57% of carbapenem-resistantK. pneumoniae isolates co-harboring carbapenemase genes were genetically related strains. This study revealed a high prevalence of intestinal carriage of carbapenem-resistant K. pneumoniae. Therefore, implementing effective screening and diagnostic measures, and focusing on antimicrobial stewardship are essential to preventing the spread of these resistant strains and minimizing the risk they pose to premature infants.

A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae

BackgroundThis study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP).MethodsThe data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC–MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro.ResultsA total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the “enzyme” group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem.ConclusionsCRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

Prevalence and molecular determinants of carbapenemase-producing Klebsiella pneumoniae isolated from Jazan, Saudi Arabia

Introduction: The World Health Organization (WHO) designated Carbapenem-resistant Enterobacterales (CRE), formerly Enterobacteriaceae, among the global priority list of antibiotic-resistant bacteria. The rate of CRE in Arabian countries, including Saudi Arabia has increased. Here, we report the prevalence of carbapenemase-producing Klebsiella pneumoniae (CPKP) in the Jazan region, a southern coastal province of Saudi Arabia. Methodology: Eighty-six non-repetitive clinical isolates of K. pneumoniae that showed resistance to at least one of the carbapenem drugs were collected from three tertiary hospitals in the Jazan region from March 2020 to April 2021. The identification and antimicrobial susceptibility testing (AST) of isolates were performed using various automated systems. Molecular detection of carbapenemase genes was conducted using a multiplex PCR. Results: Out of the 86 tested CRKP isolates, 64 (74.4%) were carbapenemase-producing isolates. The blaOXA-48 gene was the most predominant carbapenemase gene, detected in 65.1% (n = 56) of isolates. The blaNDM gene was detected in only 9.3% (n = 8) of isolates; three were found to be co-harbored with blaVIM. Interestingly, one isolate of CRKP was found to have carbapenemase genes (blaNDM, blaVIM and blaKPC), which was associated with COVID-19 patient. Conclusions: The incidence of carbapenemase-producing K. pneumoniae in Jazan hospitals seemed to be high, confirming the continued prevalence of carbapenem resistance in Saudi Hospitals. We report K. pneumoniae strain with triple carbapenemase genes in southern Saudi Arabia. The emergence of such an isolate could threaten patients and healthcare workers and requires great attention to rapid interventions to avoid further dissemination, particularly during the COVID-19 pandemic.

Co-occurrence of Carbapenemase Genes blaNDM, blaVIM, blaKPC and blaOXA-48 in Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa, a gram-negative bacterium, is notorious for its innate resistance to many antibiotics. Carbapenems are broad-spectrum antibiotics often used to treat severe P. aeruginosa infections. However, the emergence and proliferation of carbapenem-resistant P. aeruginosa (CRPA) strains have become a grave global health concern. This study examined the co-occurrence of four major carbapenemase genes, namely blaNDM, blaVIM, blaKPC, and blaOXA-48, in clinical isolates of P. aeruginosa. Using standard microbiological methods,150 P. aeruginosa clinical isolates were collected and identified, and antimicrobial susceptibility testing was conducted following Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) with gene-specific primers was used to detect the presence of carbapenemase genes. Among the 150 P. aeruginosa clinical isolates, 62(41.3%) were found to be carbapenem-resistant. The most detected carbapenemase genes were blaKPC (49%), blaNDM (31%), blaOXA-48 (22%), and blaVIM (9%). Notably 13(12.9%) isolates carried two carbapenemase genes. The combination of blaKPC and blaNDM genes was found in eight isolates, two isolates carried blaKPC and blaVIM, and three isolates carried blaOXA-48 and blaNDM. Four isolates (6.5%) harbored three carbapenemase genes. Co-occurrence of blaNDM, blaVIM, blaKPC, and blaOXA-48 was observed in four isolates (2.8%). Our findings highlight the alarming prevalence of carbapenemase genes, particularly blaNDM and blaKPC, in clinical isolates of P. aeruginosa. The co-occurrence of multiple carbapenemase genes in the same isolate raises concerns about the potential for horizontal gene transfer and dissemination of multidrug-resistant P. aeruginosa strains in clinical settings. Further research is needed to elucidate the molecular mechanisms underlying the co-occurrence of carbapenemase genes and their impact on the clinical outcomes of P. aeruginosa infections. Urgent measures, such as enhanced surveillance, infection control protocols, and antibiotic stewardship programs, are imperative to combat the emergence and spread of CRPA strains.

Multiplex lateral flow test strip for detection of carbapenemase genes using barcoded tetrahedral DNA capture probe-based biosensing interface.

Carbapenem-resistant Enterobacterales pose significant global health challenges due to their rapid spread and ability to hydrolyse various beta-lactam antibiotics. Rapid tests for these carbapenemase genes are crucial to ensure appropriate prescription administration and infection control. In this study, we developed a rapid visual nanodiagnostic platform for multiplexed detection of carbapenemase genes using a lateral flow strip. The nanodiagnostic strip was designed with separate barcoded DNA tetrahedrons for the blaKPC and blaNDM genes. These tetrahedrons were distributed on a nitrocellulose membrane at two different test lines as capture probes. When tested against a panel of carbapenemase genes, the tetrahedral probes captured single-stranded amplicons of asymmetric PCR via strand hybridisation. The amplicons acted as bridging elements, binding the DNA-modified gold nanoparticles to the test line of the strip, resulting in clear visual readouts specific to the blaKPC and blaNDM genes. By employing barcoded tetrahedrons and asymmetric PCR in conjunction with the lateral flow strip, a single diagnostic test enabled the detection of multiple carbapenemase genes. The test yielded results as low as 0.12 fM for blaKPC and 0.05 fM for blaNDM within 75min. Furthermore, the strip effectively identified specific carbapenemase genes in clinical isolates using real-time PCR, antibody-based lateral flow systems for carbapenemase detection, and carbapenemase phenotype experiments. Thus, the strip develop has a high potential for testing blaKPC and blaNDM genes in practice.

Molecular characterization of Carbapenem-resistant Escherichia coli isolates from sewage at Mulago National Referral Hospital, Kampala: a cross-sectional study

BackgroundEscherichia coli (E. coli) is one of the most frequent causes of fatal bacterial infections affecting both humans and animals. The resistance to Carbapenems is mainly associated with enzyme-mediated resistance mechanism, through the acquisition of Carbapenemase genes. In Uganda, no studies have been done to detect presence of Carbapenem-resistant E. coli in sewage. We therefore carried out a study to characterize Carbapenem-resistant E. coli from sewage from Mulago National Referral Hospital.Methods and resultsIn this cross-sectional study, a total of 104, sewage samples were aseptically collected, cultured on MacConkey agar supplemented with Meropenem 1 µg/ml with other standard microbiology methods to screen for Carbapenem-resistant E. coli (CREC). Antimicrobial susceptibility testing was performed on the CREC, using Imipenem (10 mg/disc) and Meropenem (10 mg/disc), Carbapenem drugs readily available on market. Multiplex PCR was performed on selected Carbapenem-resistant and susceptible isolates to detect Carbapenemase genes. Later the isolates were pathotyped for virulence genes that included pathogenicity islands (PAIs) and phylogenetic markers. The results showed that the Carbapenem-resistant E. coli isolates were more resistant to Meropenem (64%) than Imipenem (60%). KPC gene was the most predominant (75%), followed by NDM gene (30%) while no OXA-48, IMP-1, and IMP-2 genes were detected. Pathotyping of virulence genes showed presence of eae gene, as the most predominant (40%), followed by elt gene (25%) and negative for stx and aggR genes. For PAI markers, only the PAI IV536 gene was detected at 10%. Then, pathotyping of the phylogenetic markers was present in 85% of the typed isolates with yjaA gene the most abundant (60%) while both chuA and TSPE4.C2 were detected in 5% of the isolates.ConclusionBoth pathogenic and non-pathogenic Carbapenem-resistant E. coli strains are present in the sewage of Mulago National Referral Hospital in Uganda.