Meropenem Disc Research Articles

Significance of Modified Hodge Test in Carbapenemase Detection: A Brief Insight

Neonatal sepsis is one of the leading causes of neonatal deaths. A. baumannii-calcoaceticus is the most notorious bacterial agent. Carbapenems are the most important antibiotics and modified Hodge Test is considered as important phenotypic method for observing Carbapenemase production. Objective: To evaluate the efficacy rate of Modified Hodge Test, for detection of Carbapenem resistance. Methods: A cross sectional study was conducted at department of pathology, Nishtar Medical University, Multan from August 2023 to September 2023. The blood samples of suspected cases of sepsis were collected and after isolation of Acinetobacter baumannii sensitivity of multiple antibiotics were checked by disc diffusion method. Carbapenem resistance was re-evaluated by Modified Hodge Test using Meropenem disc (10 µg). All data were entered and analyzed by SPSS version 23.0. Results: Total samples of neonatal sepsis were 182. 83 (45.6%) were culture positive for bacterial growth. Among the positive samples 26 (31.3%) were isolated as Acinetobacter baumannii. Kirby-Bauer disc diffusion method was used to check sensitivity of multiple antibiotics including Carbapenems. Out of 26 Acinetobacter isolated samples, 16 were found to be Carbapenem resistant by this method. Modified Hodge test was used to re-confirm Carbapenem resistance. Out of 16 Meropenem resistant cases this phenotypic test only detected 5 cases (31.25%). Conclusions: Staphylococcus aureus followed by Acinetobacter baumannii were isolated predominantly and Carbapenem resistance has markedly increased. In contrast to a study conducted in 2010 in Pakistan on MHT effectiveness where effectiveness of MHT for Carbapenemase detection was satisfactory, our results reveled that some other techniques should be introduced for Carbapenemase detection as Modified Hodge test did not give satisfactory results

Characterizing carbapenemase production in Enterobacterales through combined disk test and genetic profiling

Objectives: Carbapenem-resistant Enterobacterales (CRE) are a major organism among the critical group of drug-resistant bacteria and are associated with high morbidity and mortality. With limited treatment options, the detection and characterization of carbapenemase are important for appropriate management. This study aims to characterize carbapenemase produced by Enterobacterales using a combined disk test and molecular profiling. Materials and Methods: All CRE isolated from various clinical samples were included in the study. Carbapenemase production was characterized by observing synergy on combining meropenem disk with beta-lactamase inhibitors such as phenylboronic acid, ethylenediaminetetraacetic acid, and cloxacillin, following which genetic profiling was done using multiplex polymerase chain reaction. Statistical analysis: Statistical analyses were done using the Statistical Package for the Social Sciences Statistics and Microsoft Excel. The data were presented in tables, charts, and graphs to elucidate the findings comprehensively. Results: Out of 445 Enterobacterales isolated, 104 (23.4%) were carbapenem-resistant. The most common CRE isolated was Klebsiella pneumoniae (62 out of 104), followed by Escherichia coli (40 out of 104), and two out of 104 CRE isolates were Enterobacter species. Coproduction of NDM and OXA-48-like enzymes (39.4%) was the most common mechanism, followed by NDM alone (19.2%) and OXA-48 alone (16.3%). NDM was the most common gene detected overall, with 72 out of 104 CRE (69.2%) isolates showing its presence, followed by OXA-48 present in 63 of 104 (60.6%) isolates. Conclusions: Metallo-beta-lactamases (NDM) were the predominant type of carbapenemase gene detected among the Enterobacterales isolates, with the coproduction of NDM and OXA-48 enzymes being the most common mechanism of resistance.

Characterisation of carbapenemase-producing Gram-negative bacilli among clinical isolates in a tertiary care centre in Kerala, South India

Background: Infections caused by carbapenem-resistant Gram-negative bacteria are a cause for concern due to the limited choice of antibiotics available for their treatment. Aims, Settings and Design: This study screened multidrug-resistant (MDR) Gram-negative bacilli isolated from clinical samples over a period of one year, for carbapenem resistance and characterised them using phenotypic methods such as combined disc diffusion test (CDDT), modified Hodge test (MHT), E-test for metallo-beta-lactamase (MBL) and molecular method, PCR. Materials and Methods: Two hundred and ten MDR Gram-negative bacilli were screened for carbapenem resistance using Imipenem and Meropenem disc diffusion. These were further checked for carbapenemase production by CDDT, MHT and E-test for MBL. Those positive by E-test were subjected to PCR. Uniplex PCR for New Delhi metallo-beta-lactamase-1 was used for Escherichia coli and Klebsiella pneumoniae , and multiplex PCR for Imipenemase and Verona imipenemase was used for Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Results: Twenty-three (11%) isolates were found to be carbapenem-resistant and included E. coli (six) K. pneumoniae (three), P. aeruginosa (five) and A. baumannii (nine). Seventeen (74%) isolates were positive by phenotypic methods and were subjected to PCR. Out of eight Enterobacteriaceae isolates subjected to PCR, all were positive for bla NDM gene. All were negative for bla KPC gene. All five A. baumannii isolates subjected to PCR were found to contain bla VIM gene. Two out of four P. aeruginosa isolates were positive for bla IMP , one was positive for bla VIM gene. One P. aeruginosa isolate was positive for both bla IMP and bla VIM gene. Conclusions: In view of the increasing resistance of Gram-negative bacilli to carbapenems, rational use of antibiotics needs to be emphasised.

Characterizing carbapenemase production in Enterobacterales through combined disk test and genetic profiling

: Carbapenem-resistant (CRE) are major organisms amongst the critical group of drug-resistant bacteria and are associated with high morbidity and mortality. With limited treatment options, the detection and characterization of carbapenemase is important for appropriate management. This study aims to characterize carbapenemase produced by using combined disk test and their molecular profiling. : All carbapenem-resistant isolated from various clinical samples were included in the study. Carbapenemase production was characterized by observing synergy on combining meropenem disk with beta-lactamase inhibitors like phenylboronic acid, EDTA, and cloxacillin, following which genetic profiling was done using multiplex PCR.: Out of 445 isolated, 104 (23.4%) were carbapenem-resistant. The most common CRE isolated was (62 out of 104) followed by (40 out of 104) and 2 out of 104 CRE isolates were species. Co-production of NDM and OXA-48-like enzymes (39.4%) was the most common mechanism followed by NDM alone (19.2%) and OXA-48 alone (16.3%). NDM was the most common gene detected overall with 72 out of 104 CRE (69.2%) isolates showing its presence, followed by OXA-48 present in 63 of 104 (60.6%) isolates.: Metallo-beta-lactamases (NDM) was the predominant type of carbapenemase gene detected among the isolates, with the co-production of NDM and OXA-48 enzymes being the most common mechanism of resistance.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal

BackgroundKlebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae.Materials and MethodsA totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 μg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene.ResultOf the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates.ConclusionSince the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Reliability of a Screening Method Using Antibiotic Disks to Detect Carbapenemases in Glucose-Nonfermenting Gram-Negative Microorganisms From Clinical Samples of a Regional Hospital in Southeastern Spain.

Infections by glucose-nonfermenting gram-negative bacilli (NFGNB) pose a major public health problem due to multiresistance to beta-lactam antibiotics, especially plasmid-borne carbapenemases. Their detection by microbiology laboratories is challenging, and there is a need for easy-to-use and reliable diagnostic techniques. Our objective was to evaluate an in-house screening method to presumptively detect carbapenemases in NFGNB in a simple and clinically useful manner. The study included 175 NFGNB isolates from urinary, respiratory, and rectal samples. In a triple assay, isolates were incubated at 37°C for 24 h on three solid-culture media: MacConkey II Agar, 5% Sheep Blood Columbia Agar and Mueller Hinton II Agar; meropenem (MEM) and cefepime (FEP) disks were employed for screening. Studies were then performed on the inhibition halo diameter, scanning effects, and the appearance of mutant colonies, which were compared with those observed using the colorimetric Neo-Rapid CARB Kit and immunochromatography (NG5-Test Carba and K-Set for OXA-23). Receiver operating characteristic curves were constructed for these data. Carbapenemases were expressed by 79/175 (45.1%): 19 Pseudomonas aeruginosa and 60 Acinetobacter baumannii. Optimal inhibition halo diameter cutoffs to detect this resistance on 5% sheep blood agar were as follows: 6 mm (MEM) and 6.5 mm (FEP) for P. aeruginosa (in the absence of scanning effects and mutations) and 10.5 mm (MEM) and 16 mm (FEP) for A. baumannii (even in the presence of scanning effects). The combined utilization of MEM and FEP antibiotic disks in 5% sheep blood agar, measuring their inhibition haloes, offers an effective method to predict the presence of carbapenemases as resistance mechanism in P. aeruginosa and A. baumannii.

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador

IntroductionCarbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022.MethodsNinety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC,blaNDM,blaOXA-48,blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes.ResultsCarbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases.ConclusionThe epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Detection of carbapenemase producing enterobacterales using the Modified Hodge Test from clinical isolates in Colombo South Teaching Hospital and Sri Jayewardenepura General Hospital, Sri Lanka in 2017

Introduction: Enterobacterales is a large family of Gram-negative bacilli including many pathogens. Carbapenemase producing Enterobacterales (CPEs) have emerged as a global threat. This study was conducted to detect carbapenemase production by Enterobacterales isolates from clinical specimens and to correlate the occurrence of CPE with age, gender, and duration of hospital stay of the patients included in the study.Methods: A descriptive cross-sectional study was carried out using 120 consecutive, non-repetitive clinical isolates identified as Enterobacterales by the microbiology laboratories of Colombo South Teaching Hospital and Sri Jayewardenepura General Hospital. The demographic data of the patients was gathered and used to analyse the correlation with CPE occurrence. All isolates were identified up to species level using API 20E kits. Screening for detection of carbapenemase was carried out using meropenem, imipenem, and ertapenem disks. The Modified Hodge Test (MHT), which is one of the suggested procedures to determine carbapenemase production, was performed to identify carbapenemase producing isolates.Results: Of the120 isolates, 14 (11.7%) were resistant to at least one of the carbapenems tested. MHT detected carbapenemase production in ten (8.3%) isolates. The majority of these isolates were Klebsiella sp. (6; 60.0%). Of carbapenemase producing isolates, six were from urine specimens (60%). Eight (80.0%) of the CPE harbouring patients were males and eight (80.0%) were aged above 50 years. The mean duration of hospital stay of the patients was 7.2 days (±SD 5.65days).Conclusion: The proportion of CPE was 8.3% according to this study and emphasises the importance of conducting more studies on the prevalence of CPE in Sri Lanka.

Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 μg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Antimicrobial resistance including extended spectrum beta lactamases (ESBL) and carbapenemase in feacal <em>E. coli</em> isolates from bats

Antimicrobial resistance (AMR) is one of the major causes of rising human mortality in hospitals. Although mitigation of AMR is challenging, one health approach has shown success. Bats, a key component in the ecosystem, contribute to about 20% of the world’s entire mammalian population with > 1200 species. Bats found both in rural and urban environments coexisting with the human population play a significant role in agriculture. Although AMR with significant importance has been reported in bats in some countries, a dearth of literature including dissemination of phenotypic antimicrobial resistance in bats Sri Lanka. Therefore, this study was undertaken to analyse the phenotypic antimicrobial resistance of E. coli isolated from bats in the Kandy suburbs in Sri Lanka in June – December 2022 when the worst economic crisis prevailed in the country. The fresh faecal samples were collected from three identified sites in Kandy suburbs. Faecal samples were cultured in MacConkey agar, sub-cultured in blood agar and the identification of E. coli was carried out by conventional methods. A total of 251 E. coli isolates were screened for antimicrobial susceptibility testing by the disk diffusion test. Cefotaxime and meropenem disks were used to determine ESBL and carbapenem resistance respectively. Resistance to carbapenem was observed in E. coli at 8.9%, 7.5% and 7.3% and to extended spectrum beta lactamases (ESBL) at 26.9%, 38.9% and 39.8% in the three sites where samples were collected. Over 50% of frequency was observed for trimethoprim, ampicillin and sulfa trimethoprim combination. The CTX-M was found and NDM-1 was not found by PCR similar to the finding reported previously. Surveillance of AMR in multiple sectors for baseline information on each species in the ecosystem is vital for the interpretation of resistant frequencies. However, the role of bats in the dissemination of AMR determinants needs to be further investigated.

Survey for Carbapenem Resistant Klebsiella Pneumoniae among Patients Attending Aminu Kano Teaching Hospital, Kano, Nigeria

Carbapenem resistant Klebsiella pneumoniae is becoming a more significant pathogen because of the grave clinical risk it poses which affects patient’s treatment outcome. This study aimed to survey for carbapenem resistant Klebsiella pneumoniae (CRKP) among patients attending Aminu Kano Teaching Hospital, Kano State, Nigeria. A total of 600 clinical samples including urine, sputum and swabs were collected and screened. Klebsiella pneumoniae were isolated and confirmed using culture, microscopy and biochemical test according to standard microbiological techniques. Phenotypic carbapenem resistant and carbapenemase production were detected using meropenem discs diffusion method and modified Hodge test respectively. Genotypic carbapenemase productions were confirmed by detecting the presence of blaKPC genes using PCR and Gel electrophoresis. Antibiotics susceptibility profiles of the CRKP isolates were evaluated using disc diffusion method and interpreted using CLSI protocols. The study revealed the incidence of Klebsiella pneumoniae infection as 14% with the highest occurrence in sputum (18%) and the lowest in swab (10%). Of the total number of isolated K. pneumoniae, 14.3% were found to be carbapenem resistant. Up to 75% of CRKP isolates were found to produce carbapenamase and harbor blaKPC genes. Antibiotic susceptibility profile of these isolates revealed colistin and tigecycline as the most active antibiotics in vitro (92%).The isolates were found to be generally resistant to cefepime, ceftriaxone, cefuroxime, cotrimoxazole and ciproflaxin (100%). Up to 8% of the isolates were sensitive to Gentamicin and Nalidixic acid, 25% to Nitrofurantoin, 50% to Minocycline and 17% to Amikacin. This study verifies the existence of carbapenem resistant Klebsiella pneumoniae isolates that are highly resistant to other antibiotics in patients attending AKTH Kano which is an indication of increase in drug resistance. This requires the need for newer tactics in infection control to prevent the spread of carbapenem resistant isolates.

Usefulness of inclusion of ertapenem and temocillin screening breakpoints in the EUCAST rapid antimicrobial susceptibility testing (RAST) for rapid detection of OXA-48-producing Klebsiella pneumoniae directly from positive blood cultures.

The aims of this study were: (i) to assess the ability of the meropenem screening breakpoint as part of the screening rapid antimicrobial susceptibility testing (sRAST) of EUCAST for the detection of OXA-48 carbapenemase-producing Klebsiella pneumoniae directly from positive blood cultures (BCs); and (ii) to evaluate the inclusion of ertapenem and temocillin discs into the sRAST to enhance the detection of OXA-48-producing isolates. BC bottles were spiked with a total of 117 K. pneumoniae isolates, including 77 previously characterized OXA-48 producers and 40 non-OXA-48 producers. Disc diffusion assays were directly performed from positive BCs with meropenem (10 µg), ertapenem (10 µg) and temocillin (30 µg) discs, and inhibition zones were manually measured after 4, 6 and 8 h of incubation. The screening cut-off values of sRAST were applied to evaluate their capability in detecting OXA-48-producing isolates. Receiver operating characteristic curves were constructed to illustrate the performance efficacy of the disc diffusion assays to detect OXA-48 producers. The meropenem cut-off values of sRAST only detected 90.91% of the OXA-48-producing isolates after 6 and 8 h of incubation. With the proposed cut-off points for ertapenem [<19 mm (4/6 h) and <20 mm (8 h)] and temocillin [<10 mm (4 h) and <11 mm (6/8 h)], all OXA-48-positive isolates were detected without any false-positive results at any reading time. In healthcare settings with a high prevalence of OXA-48 producers, the inclusion of ertapenem and temocillin discs in the sRAST procedure may improve the detection of OXA-48-producing K. pneumoniae isolates directly from positive BCs, providing reliable results after only a 4 h incubation period.

Diagnostic Efficacy of Carba NP Strip Test for Carbapenemase Detection

Abstract Background Bacterial resistance to carbapenems is in the focus of attention because it envoys a time without effective antibiotics. Carbapenemase enzyme production is the main mechanism of resistance exhibited by carbapenem resistant organisms. Rapid detection of carbapenemase producing bacteria is important for initiation of treatment and infection control measures. Aim of the Work Determination of the diagnostic performance of the Carba NP strip test for the detection of carabapenemase producing gram negative bacteria. Materials and Methods The study included a total of 50 gram negative clinical isolates. All isolates were subjected to: Carba NP tube test, Carba NP strip test, and meropenem disc diffusion. All techniques were performed according to Clinical and laboratory standard institute (2018). Results Compared to the results of the meropenem (10 ug) disc diffusion method, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba NP tube test was found to be 77.5%, 100%, 100% and 52.6 % respectively. On the other hand, the Carba NP strips test was found to have 82.5% sensitivity, 100% specificity, 100% PPV and 58.8% NPV. Conclusion The Carba NP test performed by use of the modified strip method was found to be a simple and inexpensive method that can be used for the rapid detection of carbapenemase producing gram negative bacteria with high sensitivity and specificity.

Associations between β-Lactamase Types of Acinetobacter baumannii and Antimicrobial Resistance.

Background and objective: Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen that not only possesses intrinsic resistance to many classes of antibiotics, but is also capable of rapidly developing antimicrobial resistance during treatment. The aim of this study was to determine the characteristics of resistance of A. baumannii strains to β-lactams and other tested antibiotics, to evaluate the associations between the phenotypes of resistance to β-lactams and other tested antibiotics, and to evaluate the changes in antibiotic resistance of A. baumannii strains over 5 years by comparing the periods of 2016-2017 and 2020-2021. Materials and methods: A total of 233 A. baumannii strains were isolated from different clinical specimens of patients treated at the Hospital of Lithuanian University of Health Sciences in 2016-2017 (n = 130) and 2021-2022 (n = 103). All clinical cultures positive for A. baumannii were analyzed. The type of β-lactamase was detected by phenotypic methods using ESBL plus AmpC screen disk tests and the combination meropenem disk test. Results: In both periods, all A. baumannii strains were resistant to ciprofloxacin; resistance to carbapenems, piperacillin/tazobactam, gentamicin, and tobramycin was noted in more than 80% of strains. A comparison of two periods showed that the percentages of A. baumannii strains producing two or three types of β-lactamases were significantly greater in 2021-2022 than in 2016-2017 (94.2% and 5.8% vs. 17.7% and 2.3%, respectively, p < 0.001). Isolates producing two or three types of β-lactamases were more often resistant to tigecycline, tetracycline, and doxycycline than strains producing one type of β-lactamase (p < 0.001). Conclusions: The frequency of isolation of A. baumannii strains producing two different types of β-lactamases (AmpC plus KPC, AmpC plus ESBL, or ESBL plus KPC) or three types of β-lactamases (AmpC, KPC, and ESBL) and the resistance rates to ampicillin/sulbactam, tigecycline, tetracycline, and doxycycline were significantly greater in 2020-2021 as compared with 2016-2017. The production of two or three types of β-lactamases by A. baumannii strains was associated with higher resistance rates to tetracyclines.

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

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

The optimized carbapenem inactivation method for objective and accurate detection of carbapenemase-producing Acinetobacter baumannii.

The modified carbapenem inactivation method (mCIM) recommended by the Clinical and Laboratory Standards Institute is not applicable for detecting carbapenemases in Acinetobacter baumannii. Four currently reported phenotypic detection methods, namely, the modified Hodge test, the mCIM, the adjusted mCIM, and the simplified carbapenem inactivation method (sCIM), did not perform well in our 90 clinical A. baumannii isolates. Thus, the minimal inhibitory concentrations (MICs) of carbapenems and the existence and expression of carbapenemase-encoding genes were detected to explain the results. According to the E-test, which was more accurate than the VITEK 2 system, 80.0 and 41.1% were resistant to imipenem (IPM) and meropenem (MEM), respectively, and 14.4 and 53.3% exhibited intermediate resistance, respectively. Five β-lactamase genes were found, of which blaOXA-51-like, blaTEM, and blaOXA-23-like were detected more frequently in 85 non-susceptible strains. The expression of blaOXA-23-like was positively correlated with the MIC values of IPM and MEM. Therefore, an improved approach based on the mCIM, designated the optimized CIM (oCIM), was developed in this study to detect carbapenemases more accurately and reproducibly. The condition was improved by evaluating the factors of A. baumannii inoculum, incubation broth volume, and MEM disk incubation time. Obvious high sensitivity (92.94%) and specificity (100.00%) were obtained using the oCIM, which was cost-effective and reproducible in routine laboratory work.

Replacement of the Double Meropenem Disc Test with a Lateral Flow Assay for the Detection of Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa in Clinical Laboratory Practice.

The prompt detection of carbapenemases among Gram-negative bacteria isolated from patients' clinical infection samples and surveillance cultures is important for the implementation of infection control measures. In this context, we evaluated the effectiveness of replacing phenotypic tests for the detection of carbapenemase producers with the immunochromatographic Carbapenem-Resistant K.N.I.V.O. Detection K-Set lateral flow assay (LFA). In total, 178 carbapenem-resistant Enterobacterales and 32 carbapenem-resistant Pseudomonas aeruginosa isolated in our hospital were tested with both our established phenotypic and molecular testing procedures and the LFA. The Kappa coefficient of agreement for Enterobacterales was 0.85 (p < 0.001) and 0.6 (p < 0.001) for P. aeruginosa. No major disagreements were observed and notably, in many cases, the LFA detected more carbapenemases than the double meropenem disc test, especially regarding OXA-48 in Enterobacterales and VIM in P. aeruginosa. Overall, the Carbapenem-Resistant K.N.I.V.O. Detection K-Set was very effective and at least equivalent to the standard procedures used in our lab. However, it was much faster as it provided results in 15 min compared to a minimum of 18-24 h for the phenotypic tests.

The Prevalence of Colonization with Carbapenem-resistant Enterobacteriaceae, Escherichia coli, Klebsiella and Enterobacter, and Related Risk Factors in Children

Background: Carbapenems are broad-spectrum antibiotics used to treat the family of gram-negative Enterobacteriaceae, especially those that are resistant to first-line antibiotics. Because these drugs are usually prescribed as the last line of treatment, resistance to these antibiotics carries irreparable risks to treatment systems, and screening high-risk individuals in medical centers and using infection control measures are critical strategies for eliminating them. Objectives: We investigated the prevalence of colonization of different strains of Enterobacteriaceae, Klebsiella, Enterobacter, and Escherichia coli and their risk factors in hospitalized children. Methods: In this descriptive cross-sectional study, stool samples were taken from patients during the first 48 hours of hospitalization in a tertiary children’s hospital and were cultured on Makcanki culture medium or EMB. Cultured Enterobacteriaceae samples were transferred to Müller-Hinton agar medium, and their antibiotic susceptibility was evaluated with meropenem and imipenem discs by disc diffusion method. In the next step, five common carbapenemase genes, including (VIM, IMP, OXA-48, NDM-1, and SPM-1) were examined by PCR method and reported accordingly. Results: Two hundred and ninety-five stool samples were examined, of which 242 (82%) samples were cultured positively with Enterobacteriaceae. The prevalence of carbapenem resistance was reported to be 37% among 295 samples using the phenotypic method. Resistance rates were high in patients with a history of antibiotic use, with frequent hospitalizations (more than two episodes in the last six months), and in patients with an underlying disease) malignancy, GI diseases, immunodeficiency, neurologic diseases such as cerebral palsy and epilepsy, endocrine diseases. Most of the genes found were OXA-48, followed by IMP and VIM. NDM-1 was found in 3 samples, and SPM was not found in any of the samples. In 13% of resistant samples, more than one carbapenemase gene was found. Conclusions: The results of this study showed that the frequency of carbapenem resistance in stools colonized with Enterobacteriaceae is high in our patients. On the other hand, the presence of carbapenemase genes in these bacteria, which are located on the plasmids that can be rapidly spread in the hospital environment, is an alarm for the hospital infection control committee to take preventive measures in order to prevent the spread of these bacteria in the hospital.

Antibiotic Susceptibility Patterns for Carbapenem-Resistant Enterobacteriaceae.

Carbapenem is a broad-spectrum beta-lactam antibiotic considered the last choice for the treatment of antibiotic-resistant Gram-negative bacteria. Thus, the increasing rate of carbapenem resistance (CR) in Enterobacteriaceae is an urgent public health threat. This study aimed to evaluate the antibiotic susceptibility pattern of carbapenem-resistant Enterobacteriaceae (CRE) to new and old antibiotics. In this study, Klebsiella pneumoniae, E. coli, and Enterobacter spp. were collected from 10 hospitals in Iran for one year. CRE is recognized by resistance to meropenem and/or imipenem disk after identification of the collected bacteria. Antibiotic susceptibility of CRE against fosfomycin, rifampin, metronidazole, tigecycline, and aztreonam was detected by disk diffusion method and colistin by MIC. In this study, 1222 E. coli, 696 K. pneumoniae, and 621 Enterobacter spp. were collected from 10 hospitals in Iran in one year. Fifty-four E. coli (4.4%), 84 K. pneumoniae (12%), and 51 Enterobacter spp. (8.2%) were CRE. All CRE strains were resistant to metronidazole and rifampicin. Tigecycline has the highest sensitivity on CRE and levofloxacin for Enterobacter spp. Tigecycline showed an acceptable effectiveness rate of sensitivity on the CRE strain. Therefore, we suggest that clinicians consider this valuable antibiotic to treat CRE.

A Rapid and Easy Method of MALDI Biotyper Antibiotic Susceptibility Test Rapid Assay To Provide Early Meropenem Susceptibility Profile in Enterobacterales.

Antimicrobial susceptibility testing (AST) that can provide faster results is necessary. The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) can provide early AST results but still needs to be simplified in order to facilitate its execution by microbiology laboratories. The aim of this study was to evaluate an adaptation of MBT-ASTRA. Isolates of Enterobacterales were tested for meropenem susceptibility by MBT-ASTRA using a solution prepared from meropenem disks and performing a manual spectrum analysis. The relative growth (RG) was calculated for each isolate, and a cutoff value was established to determine the susceptibility profile of the isolates. Results of the adapted method were compared with the standard susceptibility method (broth microdilution). An adapted method of MBT-ASTRA was developed. The RG cutoff values for meropenem were ≤0.1510 for susceptibility and >0.6272 for resistance, presenting 95.65% categorical agreement, with 2.9% (2/69) minor discrepancy and 3.23% (1/31) very major discrepancy. MBT-ASTRA can be used to provide rapid AST results with a simpler and more accessible protocol, especially regarding spectrum analysis. IMPORTANCE The simplification of the MBT-ASTRA technique, especially in spectrum analysis, can considerably allow more laboratories to rapidly determine antimicrobial susceptibility profiles.