Combined Disk Test Research Articles

Detection of Carbapenemase-Producing Enterobacterales: A Comparative Analysis of Available Phenotypic Methods

Background: Carbapenems serve as a critical last-line defence against infections from multidrug-resistant Enterobacterales. The increasing prevalence of CRE (carbapenem-resistant Enterobacterales) poses significant challenges in clinical settings. This greatly complicates the handling of gram-negative bacilli (GNB) infections and represents a significant global health threat. Aim: This study evaluates and contrasts different phenotypic techniques used to identify carbapenemase-producing Enterobacterales isolated from various clinical samples. Objectives: To compare the accuracy and reliability of Combined Disc Test (CDT) and Modified Carbapenem Inactivation Method/EDTA - Carbapenem Inactivation Method (mCIM/eCIM) in identifying Metallo-beta-lactamase (MBL)-producing Enterobacterales. Methods: All clinical specimens submitted for culture and sensitivity testing at the Department of Microbiology were analysed to isolate and identify Enterobacterales. Carbapenemase production was assessed through using the phenotypic CDT Confirmation was carried out using the mCIM and eCIM in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: During a two-year period, 254 strains of Enterobacterales were isolated. Among those, 80/254 (31.49%) isolates exhibited resistance to the Imipenem (IMP) disc. The CDT identified 30/80 (37.5%) isolates as serine carbapenemase producers and 50/80 (62.5%) isolates as MBL producers. In comparison, the mCIM/eCIM methods detected 26/80 (32.5%) isolates as serine carbapenemase producers, 48/80 (60.0%) isolates as MBL producers, and 6/80 (7.5%) isolates were tested negative for carbapenemase production. Conclusion: This study evaluates the precision and effectiveness of the mCIM/eCIM and CDT methods for identifying CRE. The combination of the mCIM and eCIM tests may be a more reliable phenotypic method for detecting carbapenemase compared to the CDT.

Prevalence of Metallo β-Lactamase Genes Among MDR Pseudomonas <i>Aeruginosa</i> Isolated from Different Sources in Baghdad

Background: Pseudomonas aeruginosa is a gram-negative apportunistic pathogen that has ability to couse different type of infections and charecterised by their high resistance to commonly used antibiotics via different stratiges. Objective: The objective of this study is to detect the prevalence rate of MβL gene among MDR Pseudomonas aeruginosa. Methods: Forty clinical isolates of Pseudomonas aeruginosa were obtained from different hospitials in Baghdad/Iraq. Their identification was confirmed by using 16s rDNA as a housekeeping gene (reference gene). Specific primers were used to detect 3 types of Metallo β-lactamase (MβL) genes vim, spm1, and imp1 genes followed by sequencing the amplified fragment which was analyzed by Geneious software. Antibiotic sensitivity test for 15 antimicrobial agents was done using the Kirby-Bauer disc diffusion method. Results: The revealed resistance pattern was as following: 100% for the combination of Trimethoprim/Sulphamethoxazole and Nitrofurantoin, 95% for Tigecycline, 82.5% for Ciprofloxacin, 67.5% for Cefepime, 60% for Levofloxacin, 57.5% for Carbenicillin, 55% for Piperacillin, 50% for Amikacin, 47.5% for Tobramycin, 45% for both Imipenem and Ceftazidime, 27.5% for piperacillin/tazobactam, 22.5% for Aztreonam and 7.5% for Colistin. More than half (55%) of isolates were positive for MβL enzymes production during phenotypically assessed by combined disc test. The MIC of meropenem ranged from 16µg/ml to 32 µg/ml. The percentage of MβL genes among the total isolates during conventional PCR was as follows: 95% for vim, 25% of the isolates harbored both vim and imp1, while spm 1 are negative in all isolates. The result of the study explained that 10 isolates from the antibiotic susceptibility test were resistant to 10-14 antimicrobial agents that harbored both vim and imp1 genes. Conclusions: MβL gene could be used as a genetic marker to determine the degree of resistance in MDR P. aeruginosa which are more resistant than other isolates that have one gene of MβL responsible for break the β-lactam ring then inactivating the β-lactam antibiotic.

Evaluation of the Expression of SIM and NDM Genes in Pseudomonas Aeruginosa Isolated from Clinical Sources

Pseudomonas aeruginosa has recently been labeled a major threat to public health due to its resistance to almost all commonly used antibiotics. Many factors have been suggested for P. aeruginosa's antibiotic resistance. The goal of this study is to find out what role SIM and NDM, which are related to carbapenem resistance, play in P. aeruginosa isolates from local clinical sources. In this study, out of 110 different clinical specimens, 50 were identified as P. aeruginosa from hospitalized patients. All of the isolates were characterized based on the biochemical test and confirmed using the VitekII compact system. P. aeruginosa isolates were tested for antibiotic susceptibility using 8 antibiotics, including: amikacin, tobramycin, ciprofloxacin, levofloxacin, imipenem, meropenem, piperacillin/tazobactam, ticarcillin, and clavulanate. Susceptibility testing results revealed that every isolate was highly resistant to Piperacillin/Tazobactam, with lower resistance to Ticarcillin/clavulanate (TCC). Using the broth dilution method, the minimum inhibitory concentration (MIC) of P. aeruginosa isolates resistant to meropenem ranged from 32µg/100µl to 128 µg/100µl. The EDTA combined disc test was used to detect the ability of P. aeruginosa isolates to produce carbapenemase, and the results showed that all isolates were carbapenemase producers. Additionally, conventional PCR confirmed the identification of P. aeruginosa using 16S. Real-time PCR was adopted to assess the expression of the NDM and SIM genes in 25 of the identified P. aeruginosa isolates. The mean of gene expression results for NDM showed increased expression compared to the control sample of 1.74, while the SIM gene showed expression of 0.95. These genes, SIM and NDM in class B (which are important for resistance in Pseudomonas aeruginosa), result from chromosomal changes that mutate the membrane permeability flow pump, causing excessive expression.

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.

High-throughput screening identification of apigenin that reverses the colistin resistance of mcr-1-positive pathogens.

The plasmid-mediated gene mcr-1 that makes bacteria resistant to the antibiotic colistin is spreading quickly, which means that colistin is no longer working well to treat Gram-negative bacterial infections. Herein, we utilized a computer-aided high-throughput screening drugs method to identify the natural product apigenin, a potential mcr-protein inhibitor, which effectively enhanced the antimicrobial activity of colistin. Several assays, including a checkerboard minimum inhibitory concentration assay, a time-kill assay, the combined disk test, molecular simulation dynamics, and animal infection models assay, were conducted to verify whether apigenin enhanced the ability of colistin to fight Gram-negative bacterial infections. The results showed that apigenin improved the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae infection. Moreover, apigenin not only did not increase the toxic effect of colistin but also had the ability to effectively inhibit the frequency of bacterial resistance mutations to colistin. Studies clearly elucidated that apigenin could interfere with the thermal stability of the protein by binding to the mcr-1 protein. Additionally, the combination of apigenin and colistin could exert multiple effects, including disrupting bacterial membranes, the generation of bacterial nitric oxide and reactive oxygen species, as well as inhibiting bacterial adenosine triphosphate production. Furthermore, the addition of apigenin was able to significantly inhibit colistin-stimulated high expression levels of the bacterial mcr-1 gene. Finally, apigenin exhibited a characteristic anti-inflammatory effect while enhancing the antimicrobial activity of colistin against mcr-1-positive Escherichia coli (E. coli) infected animals. In conclusion, as a potential lead compound, apigenin is promising in combination with colistin in the future treatment of mcr-1-positive E. coli infections.IMPORTANCEThis study found that apigenin was able to inhibit the activity of the mcr-1 protein using a high-throughput virtual screening method. Apigenin effectively enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains, in vitro and in vivo. This study will provide new options and strategies for the future treatment of multidrug-resistant pathogen infections.

Assessment of in vitro activity of ceftazidime/avibactam on carbapenemase-producing Enterobacterales from Iran: An experimental study.

The prevalence of carbapenemase-producing Enterobacterales (CPE) continues to increase worldwide. Combination of β-lactam and novel β-lactamase inhibitors introduce a revolutionary treatment option for CPE. Ceftazidime/avibactam (CAZ/AVB) has been recently developed for treatment of severe infections caused by multidrug-resistant bacteria. We aimed to evaluate in vitro activity of CAZ/AVB on a collection of 85 ESBL-producing-carbapenemase negative and CPE from Iran. ESBL and carbapenemase production was phenotypically confirmed by combined disk test and modified carbapenem inactivation method respectively. The presence of clinically important carbapenemase encoding genes was examined using PCR. Susceptibility of all isolates to CAZ/AVB was determined using discs containing 30 μg ceftazidime +20 μg avibactam (AVB). Minimum inhibitory concentrations (MICs) of CAZ/AVB in 28 CPE (4 Escherichia coli and 24 Klebsiella pneumoniae) was determined by gradient diffusion method using MIC test strips (0.016-256 mg/L ceftazidime +4 mg/L AVB). All phenotypically identified ESBL positive-carbapenemase negative isolates were found to be susceptible to CAZ/AVB. Among the carbapenem resistant isolates, CAZ/AVB showed potent inhibitory activity against all OXA-48-like (MIC ranges 0.125/4-0.75/4 mg/L) and KPC positive isolates (MIC ranges <0.016/4-0.19/4 mg/L). However, AVB could not restore the activity of ceftazdime against isolates producing metallo-β-lactamases (MLBs) including VIM, NDM (MIC > 256/4 mg/L) and IMP (MIC > 8/4 mg/L). Our data highlighted the excellent in vitro performance of CAZ/AVB against ESBL-producing and CPE suggesting that this combination can efficiently be used as therapeutic option for management of CPE infections particularly in regions with high prevalence of KPC and/or OXA-48-like positive but MBL-negative Enterobacterales.

Extended-spectrum beta-lactamase and carbapenemase producing Klebsiella Pneumoniae isolated from patient with suspected urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infections in humans and serious health problem in many parts of the world. Carbapenem resistant Enterobacteriaceae (CRE) are increasingly reported from healthcare facilities in Nepal and around the world. This study is carried out with an objective to assess the Prevalence of ESBL and carbapenemase producing Klebsiella pneumoniae isolate from patient with suspected of UTI. Total 880 urine sample was collected during 6-month period and processed and identified by using conventional microbiological procedure and biochemical test. ESBL screening isolates were done by using Ceftazidime and Ceftazidime with clavulanic acid. Production of ESBL was determine by combination Disc Test. For the confirmation of carbapenemase producing K. pneumoniae Modified Hodge Method, Carbapenem Inactivation Method and EDTA Impregnated Disc test (MBL) was performed. Out of total sample, 9.30% (82) showed significant growth. Prevalence of UTI seen more in male as compare to female patients. Out of positive isolates, 37.8% were K. pneumoniae. K. pneumoniae were resistance with Ceftriaxone (54.8%) followed by 51.61% Ceftazidime, Cotrimoxazole. Multi drug resistance (MDR) was found to be 54.83%. Out of 31 isolates, 10(32%) were confirmed as ESBL positive by Combination Disc Assay. 14 isolates were screened as carbapenemase producers but 2(14.28%) showed positive through Modified Hodge Method, 4(28.57%) Modified Carbapenem Inactivation Method and 7(50%) EDTA impregnated Disc Test (MBL). Analytically, the sensitivity of MHT, CIM, and MBL tests was 42.81%, 58.10%, and 76.96%, respectively, all with 100% specificity. Colistin was the drug of choice, with 93.54% of isolates showing sensitivity to it. Early detection of ESBL and MBL-producing isolates is crucial for reducing mortality rates and preventing the intra-hospital spread of these strains.

Prevalence of metallo-beta-lactamase in clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis in Benin City, Nigeria

Carbapenems are the prime choice of treatment for severe cases of infections caused by Multi-Drug-Resistant Pseudomonas aeruginosa and Proteus mirabilis. Nevertheless, Metallo-Beta-Lactamase (MBL) production by these organisms has led to carbapenem resistance which is a global threat. This study aimed to determine the prevalence and antimicrobial susceptibility profile of MBL in clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis in Benin City, Nigeria. 354 non monotonous clinical isolates of Pseudomonas aeruginosa (282) and Proteus mirabilis (72) used were obtained from various clinical samples from tertiary hospitals in Benin City. Identification of these isolates were done using standard microbiological techniques. Antimicrobial susceptibility test was performed using Kirby-Bauer disk diffusion method. MBL production was detected using Imipenem Ethylene-Diamine-Tetra-Acetic Acid combined disc test method. Of the total 354 clinical isolates tested, 115 (32.48%) were MBL and the prevalence of this resistant isolates was significantly higher in Pseudomonas aeruginosa (46.8%) compared to Proteus mirabilis (P=0.0001). Among the metallo-beta-lactamase producing isolates of Pseudomonas aeruginosa higher prevalence were reported from Pleural fluid and cerebrospinal fluid samples with 6 (100%) and 3 (100%) respectively. This difference was statistically significant (P=0.0001). Susceptibility testing showed that isolates that produced beta-lactamase demonstrated poorly against cephalosporin, amoxicillin-clavulanate, gentamicin and floroquinones than non-beta- lactamase producers. A prevalence of 46.8% was reported for MBL producing Pseudomonas aeruginosa and 12.5% was reported for MBL producing Proteus mirabilis. Isolates that produced the MBL enzyme were more resistant to antibacterial agents. Measures to control and curb the spread of MBL producing clinical isolates are highly advocated

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.

Analysis of mcr family of colistin resistance genes in Gram-negative isolates from a tertiary care hospital in India.

Colistin serves as the drug of last resort for combating numerous multidrug-resistant (MDR) Gram-negative infections. Its efficacy is hampered by the prevalent issue of colistin resistance, which severely limits treatment options for critically ill patients. Identifying resistance genes is crucial for controlling resistance spread, with horizontal gene transfer being the primary mechanism among bacteria. This study aimed to assess the prevalence of plasmid-mediated mcr genes associated with colistin resistance in Gram-negative bacteria, utilizing both genotypic and phenotypic tests. The clinical isolates (n=913) were obtained from a tertiary care center in Chennai, India. Colistin resistance was seen among Gram-negative isolates. These strains underwent screening for mcr-1, mcr-3, mcr-4, and mcr-5 genes via conventional PCR. Additionally, mcr-positive isolates were confirmed through Sanger sequencing and phenotypic testing. The bacterial isolates predominantly comprised Klebsiella pneumoniae (62.43%), Escherichia coli (19.71%), Pseudomonas aeruginosa (10.73%), and Acinetobacter baumannii (4.81%), along with other species. All isolates exhibited multidrug resistance to three or more antibiotic classes. Colistin resistance, determined via broth microdilution (BMD) using CLSI guidelines, was observed in 13.08% of the isolates studied. Notably, mcr-5 was detected in K. pneumoniae in PCR, despite its absence in Sanger sequencing and phenotypic tests (including the combined-disk test, colistin MIC in the presence of EDTA, and Zeta potential assays). This finding underscores the importance of employing multiple diagnostic approaches to accurately identify colistin resistance mechanisms.

Coexistence of β-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced β-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

New Delhi metallo-β-lactamases among extensively drug-resistant clinical isolates from Lahore, Pakistan.

Aim: The study determines rates of carbapenem resistance (CR) and frequency of blaNDM in multidrug-resistance (MDR) or extensivedrug resistance (XDR), and evaluates the potential of phenotypic tests for detecting NDMproduction. Materials & methods: Singleplex PCR was used to detect blaNDM. Phenotypic tests, including combination disc test (CDST) and modified Hodge test (MHT), were evaluated for NDM production. Results: Among 338 CR isolates, 47.63% were MDR, whereas 52.36% were XDR with 53.25% carrying blaNDM. MHT was found to bediscriminativefor detecting NDM production, whereas no significant association was observed for CDST. Conclusion: The high incidence of CR and MDR and XDR isolates possessing blaNDM presents an impending threat in therapeutics. Limitations of phenotypic tests suggest better testing, including molecular detection of the enzyme.

A comparative evaluation of five phenotypic methods for identification of carbapenemase-producing Enterobacteriaceae: a modified carbapenemase detection test.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) is urgently needed to prevent their spread in healthcare settings. Here, we have evaluated the performance of the phenotypic methods for detection of carbapenemase production directly from bacterial cultures. A total of 99 clinical and rectal Enterobacteriaceae isolates were included (81 carrying known carbapenemase-encoding genes and 18 without carbapenemase production). All isolates were subjected to the five phenotypic tests including in-house Carba NP (iCarba NP), modified-Carba NP, E-Test MBL, modified Hodge test (MHT), and commercial combination disk test. Test results were read at different time points for iCarba NP and modified-Carba (1 min, 5 min, 15 min, 1 h and 2 h). The sensitivity and specificity of the iCarba NP were 78.87% and 100%, respectively, whereas those of the modified-Carba NP test were 95.06% and 94.44%, respectively. False-negative results were detected in four OXA-48 isolates with the use of modified-Carba NP, whereas one non-carbapenemase isolate had false-positive results. The sensitivity/specificity was 91.30%/100% and 80.25%/83.33% for the E-Test MBL and MHT, respectively. The sensitivity and specificity of the aminophenylboronic acid synergy test were 100% and 97.94%, respectively, whereas those of the dipicolinic acid synergy test were 82.61% and 96.23%, respectively. Rapid, simple, and reliable methods are needed for laboratory detection of CPE isolates to improve the detection and surveillance of these clinically relevant pathogens in an epidemiological context. We conclude that the modified-Carba NP test can be one of the reliable tests for the prediction of carbapenemase-producing bacteria.IMPORTANCEThe emergence of carbapenem resistance among Gram-negative bacteria is a serious global health threat. Here, we investigate the performance of the five phenotypic assays against carbapenemase-producing and carbapenemase-non-producing Enterobacteriaceae. Accurate and rapid detection of CPE isolates is critically required for clinical management and treatment of infections caused by these organisms. Among the five evaluated phenotypic tests, the mCNP test presented the highest sensitivity (95.06%) and, therefore, can be considered the best test to be used as a screening phenotypic methodology.

Predominance of OXA-48 carbapenemase-producingKlebsiella pneumoniaestrains in tertiary hospital in Sarajevo, Bosnia and Herzegovina.

Klebsiella pneumoniae, a member of the Enterobacteriaceae family, demonstrates an increasing trend of resistance to carbapenems and is a common cause of both hospital- and community-acquired infections. The current study provides insights into the genetic characterization of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates circulating during 2022 in a Sarajevo tertiary hospital. Among the 87 CRKP strains analyzed, real-time polymerase chain reaction (rtPCR) results showed that 85 (97.7%) tested positive for the carbapenem resistance gene. The oxacillinase-48 (OXA-48) gene was detected in 83 (95.4%) isolates, while the Klebsiella pneumoniae carbapenemase (KPC) and the New Delhi metallo-beta-lactamase (NDM) genes were detected in one isolate each. No Verona integron-encoded-metallo-beta-lactamase (VIM) or imipenemase-metallo-beta-lactamase 1 (IMP-1) genes were found in any of the tested isolates. The multilocus sequence typing (MLST) analysis of sequence types (STs) revealed that ST101, an emerging high-risk clone exhibiting extensive drug resistance, was the most prevalent, whereas ST307 was detected in only one isolate. Phylogenetic analysis of the ten CRKP isolates indicated the presence of three clusters that could constitute an outbreak. A comparison of the results of the utilized phenotypic test (the combined-disk test [CDT]) and rtPCR showed high concordance, suggesting that the phenotypic assay may be useful for the early detection of resistance mechanisms as part of routine susceptibility testing. With the increased affordability of next-generation sequencing (NGS), its application in hospital settings has proven highly beneficial, aiding in the implementation of infection control and prevention measures. Given the significant resistance demonstrated by the CRKP isolates to most tested antibiotics, it is imperative to establish effective methods to restrict the spread of these isolates, as well as to carefully monitor the use of carbapenems in clinical practice.

Detection of Carbapenem Resistance Using the Genotypic and Phenotypic Methods in Klebsiella pneumoniae

Aim: This study aimed to detect the carbapenem resistance of the Klebsiella pneumoniae strains, isolated from clinical specimens with genotypic and phenotypic methods. Material and Methods: A total of 87 Klebsiella pneumoniae strains whose carbapenem resistance was determined by disc diffusion method were included in the study. Carbapenemase was investigated using the combined disk method and polymerase chain reaction (PCR). Results: The evaluation of the PCR results demonstrated that OXA was detected in 60 (68.9%) samples, NDM was detected in 20 (22.9%), OXA + NDM in 5 (5.7%), and KPC was detected in 1 (1.1%) out of 87 clinical samples. Carbapenemase was not detected in one specimen with the PCR method. The results were found compatible with the combined disc test results for all isolates which were detected as only OXA, NDM, and KPC type carbapenemase positive. In 5 (5.7%) strains in which the co-existence of NDM and OXA type carbapenemases was detected by PCR, the combined disc method detected only OXA type carbapenemase. Conclusion: The combined disk method is inadequate in the presence of strains that have multiple carbapenemases, and also have OXA which is the most frequently detected carbapenemase in our hospital. EUCAST recommends verification by other methods in the presence of OXA-48. Genotypic methods can be used for confirmation testing. The detections of strains with NDM, multiple carbapenemases, and the first detection of KPC were striking in the study. Monitoring the spread of these strains in the hospital will be necessary for infection control.

Molecular Identification of Carbapenem Resistance Acinetobacter baumannii causing Ventilator-Associated Pneumonia Isolated from Intensive Care Unit of Tertiary Care Hospital

Ventilator-associated pneumonia (VAP) is a serious complication in critically ill patients, significantly increasing morbidity and mortality. One concerning organism behind VAP is Acinetobacter baumannii, a multidrug-resistant bacterium has ability to evade treatment, particularly with carbapenems, the last-line antibiotics. This is especially worrisome within the confines of Intensive Care Units (ICUs) of tertiary care hospitals, hubs for high-risk patients and potential reservoirs of antimicrobial resistance. This study focused on identifying carbapenem-resistant Acinetobacter baumannii using both phenotypic and genotypic methods. In 132 isolates of Acinetobacter baumannii, we observed 96% resistance to the cephalosporins while least resistance found to colistin and tigecycline. However, a concerning 51.5% of isolates exhibited carbapenem resistance. Phenotypically confirmation of carbapenem resistance detected in 47% isolates by Combined Disc Test and 51.5% isolates by Modified Hodge Test and E-test. Genotypic analysis with RT-PCR revealed a diverse array of resistance genes: blaIMP (33.82%), blaVIM (25%), blaOXA-Group (20.58%), and blaNDM (8.82%). These findings highlight the alarming prevalence of carbapenem-resistant Acinetobacter baumannii in healthcare settings.

Metallo-Beta-Lactamase Producing Isolates of Escherichia coli and Klebsiella pneumoniae and their Resistance Profiles in Enugu, Nigeria: A Threat to Public Health

Background: A potential threat to public health is the rapidly spreading enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae which produce metallo-beta-lactamases (MBL). This study evaluated the prevalence of metallo-beta-lactamase (MBL) from clinical and non-clinical sources in Enugu Metropolis.&#x0D; Methodology: The study was conducted in the Microbiology Laboratory of the University of Nigeria Teaching Hospital, Ituku-Ozalla between October 2020 and July 2021. A total of 150 isolates including 85 and 65 isolates of Escherichia coli and Klebsiella pneumoniae respectively was recovered. Standard microbiology procedures were used to identify and characterize the isolates. Antimicrobial susceptibility was done using the Kirby-Bauer disc diffusion technique. Phenotypic detection of Metallo-beta-lactamase production was determined using Combined Disk Tests.&#x0D; Results: Imipenem resistance was detected in 22 (25.9%) isolates of E. coli and 18 (27.7%) isolates of K. pneumoniae. Of the 22 strains of E. coli that were imipenem resistant, 8 (9.4%) and 14 (16.5%) were found to be MBL producers and non-MBL respectively. Of the 18 strains of Klebsiella pneumoniae that were imipenem resistant, 10 (15.4%) were MBL producers and 8 (12.3%) were non-MBL producers. The highest prevalence of MBL was recovered from urine sources in both E. coli and K. pneumonieae. All MBL-producing isolates were multidrug resistant.&#x0D; Conclusion: The overall prevalence of MBL in this study was 12.0%. Public health is at risk due to the occurrence of metallo-beta-lactamase. Antimicrobial stewardship and the implementation of infection control strategies are required to halt the spread of these resistant bacteria in the environment. The use of antibiotics should be with utmost prudence.

Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

Phenotypic Detection of Extended Spectrum β-lactamase Producing Gram Negative Bacteria Isolated From Clinical Specimens

Background: Over the past decade, there has been a significant global rise in extended spectrum β-lactamase (ESBL) producing gram negative bacteria (GNB), posing a substantial concern. ESBLs, encoded by plasmids, are deemed threatening due to their ease of transmission between species. The Asia Pacific region has witnessed a surge in hospital-acquired infections attributed to ESBL-producing GNB, as highlighted by numerous studies. This study seeks to assess the prevalence of ESBL-producing GNB specifically within Nepal. Methods: This research, conducted at Grande International Hospital in Kathmandu, Nepal, adopted a descriptive cross-sectional approach. It spanned from July 2016 to February 2017 and encompassed patients receiving care at the hospital. The study involved the handling of samples directed to the Microbiology laboratory for Culture and sensitivity, as well as the identification of isolates and Antibiotic susceptibility testing, following established microbiological protocols. Screening for Multi-Drug resistance (MDR) and Extended Spectrum Beta-Lactamase (ESBL) was conducted for all Escherichia coli and Klebsiella pneumoniae isolates utilizing a combined Disk (CD) test method. Results: Out of 258 Escherichia coli and Klebsiella pneumoniae isolates, 42 isolates of E. coli and 21 isolates of K. pneumoniae were confirmed as ESBL producer. Conclusion: The regular surveillance of hospital-associated infections including monitoring of antimicrobial susceptibility pattern is needed for the formulation of a definite antimicrobial policy.

PREVALENCE AND CHARACTERISATION OF EXTENDED-SPECTRUM BETA-LACTAMASES AND PLASMID-MEDIATED QUINOLONES RESISTANCE IN Enterobacteriaceae ISOLATED FROM COMPANION ANIMALS

Antimicrobial resistance is a major public health concern worldwide. This study aims to determine the prevalence of Enterobacterales producing beta-lactamase (TEM, SHV, OXA) or extended-spectrum beta-lactamases (ESBL), as well as plasmid-mediated resistance to quinolones (PMQR) (qnrA, qnrB, qnrS) in companion animals from the northeast region of Romania. A total of 124 faecal samples were collected aseptically from healthy dogs attending the veterinary practice for vaccination and cultivated on Brilliance ESBL medium (Oxoid, UK). The ESBL production testing was performed using the combination disc test. The identification of Enterobacterales strains was achieved using molecular identification and based on biochemical tests. Antimicrobial susceptibility testing was performed using the disk diffusion method. Identification of genes encoding for beta-lactamase enzymes and genes encoding plasmid-mediated resistance to quinolones was performed by PCR according to the protocols previously described. After ESBL screening, 31 (31/124; 25%) extended-spectrum cephalosporin (ESC)-resistant Enterobacterales were obtained, and 67.74% (21/31) of them were confirmed as ESBL-producers. Regarding the Enterobacterales species, 27 (27/31; 87.1%) were Escherichia coli and 4 (4/31; 12.9%) strains were Klebsiella pneumoniae. Among the ESBL-producing isolates, the blaCTX-M-1 gene group was predominant (58.82%), followed by the blaCTX-M-9 group (41.18%). The blaTEM, blaSHV and blaOXA gene groups were identified in 54.83%, 29.03% and 3.22% of the analysed strains, respectively. The prevalence of PMQR genes was 22.58% and consisted only of qnrS (19.35%) and qnrA (3.22%) genes. The prevalence of ESBL strains related to the total number of analysed samples was 16.93% (21/124). The findings show a significant prevalence of ESBLs and PMQR genes in Enterobacterales strains isolated from the faeces of healthy dogs, implying that pets may pose a risk of transmitting ESBL strains to other animals or owners.