Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method

Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method Sumon Kumar Das , Nikhat Ara , Suma Mita Biswas , View ORCID Profile Afzal Sheikh doi: https://doi.org/10.1101/2024.12.30.24319797 Sumon Kumar Das 1 Department of Microbiology & Immunology, Evercare Hospital , Dhaka, Bangladesh Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nikhat Ara 1 Department of Microbiology & Immunology, Evercare Hospital , Dhaka, Bangladesh Find this author on Google Scholar Find this author on PubMed Search for this author on this site Suma Mita Biswas 1 Department of Microbiology & Immunology, Evercare Hospital , Dhaka, Bangladesh Find this author on Google Scholar Find this author on PubMed Search for this author on this site Afzal Sheikh 2 Department of Biochemistry & Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University , Dhaka, Bangladesh 3 Institute of Food Safety and Processing (IFSP), Bangabandhu Sheikh Mujibur Rahman Agricultural University , Dhaka, Bangladesh Email: * Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Afzal Sheikh For correspondence: asheikh{at}bsmrau.edu.bd asheikh{at}bsmrau.edu.bd Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Background The prompt identification of carbapenemases, particularly Klebsiella pneumoniae carbapenemases (KPC) and Metallo-β-lactamase (MBL) produced by Klebsiella pneumoniae and Enterobacteriaceae , is imperative in stemming the spread of pathogens carrying these antibiotic-resistant attributes. This retrospective study seeks to delve into the clinical and microbiological characteristics of patients affected by gram-negative bacteria producing KPC and MBL, shedding light on their profiles and impacts. Methods We analyzed 147 clinical isolates from tertiary care hospitals in Dhaka, Bangladesh. Bacterial identification and Minimum inhibitory concentration (MIC) analysis were performed using conventional culture and VITEK ® 2 compact system. Carbapenem non-susceptibility (Cns) was defined as a minimum inhibitory concentration (MIC) of ≥1 µg/ml for Enterobacteriaceae while Pseudomonas aeruginosa and Acinetobacter baumannii bacterial isolates Imipenem non-susceptibility was defined as an MIC of ≥2 µg/ml. Detection of KPC and MBL-producing isolates was performed using the disk potentiation test (DPT) method. Results All 147 clinical bacterial isolates demonstrated resistance to Imipenem (MIC 1 -≥16 µg/ml), with Klebsiella pneumoniae carbapenemase (KPC) detected in 26.53 % (39 out of 147) isolates and metallo-beta-lactamases (MBL) in 44.89% (66 out of 147) isolates. Carbapenem resistance was notably prevalent among older male patients of the “> 60” age group, with the ICU harboring the highest number of resistant isolates, primarily associated with Acinetobacter baumannii followed by Klebsiella pneumoniae . Conclusion Our findings unveiled a concerning prevalence of carbapenem resistance among clinical bacterial isolates, with notable proportions of KPC and MBL producers. The findings underscore the urgent need for effective surveillance and infection control measures to mitigate the spread of multidrug-resistant pathogens in healthcare settings. Introduction Infections caused by drug resistant gram-negative Enterobacteriaceae , notably Klebsiella pneumoniae producing Klebsiella Pneumoniae Carbapenemases (KPCs), pose significant global concerns. These organisms cause formidable challenges to clinicians due to limited antibiotic options, resulting in unfavorable clinical outcomes. Carbapenems serve as the primary treatment for severe infections induced by extended-spectrum beta-lactamases (ESBLs), given the escalating incidence of fluoroquinolone resistance among Enterobacteriaceae . As a result, carbapenems are being used more frequently in clinical settings to treat these infections ( 1 , 2 ). Klebsiella pneumoniae Carbapenemase (KPC) is not the sole carbapenemase identified in K. pneumoniae ; there are also reports suggesting the existence of metallo-beta-lactamases (MBL) with the ability to hydrolyze carbapenems ( 3 , 4 ). The horizontal transmission of carbapenemase genes, facilitated by mobile genetic elements such as self-transmissible plasmids or transposons, is a significant concern in antibiotic resistance. These elements not only carry carbapenemase genes but also harbor additional resistance determinants, leading to multidrug resistance. As a consequence, bacteria become resistant to multiple groups of antibiotics, often rendering them impervious to all available treatment options ( 5 , 6 ). The management of infections caused by carbapenemase-producing Enterobacteriaceae has become exceedingly difficult owing to their profound resistance against a broad spectrum of antibiotics. Compounding the challenge is the inherent nature of Enterobacteriaceae as members of the intestinal flora, facilitating their rapid spread and resilient persistence. Furthermore, the rampant utilization of antibiotics in both human medicine and in the food chain, coupled with their subsequent release into the environment, serves to expedite the selection and horizontal transfer of antibiotic-resistant genes or plasmids within bacterial populations ( 2 , 7 ). As MBLs are predominantly encoded within transposons and/or integrons, they possess a high propensity for dissemination to other enterobacterial strains ( 7 – 9 ). The primary reservoir for these bacteria is believed to be hospitals and other healthcare facilities, where instances of severe bacterial infections are prevalent and antibiotic usage is extensive ( 10 , 11 ). Numerous studies worldwide have investigated and assessed the efficacy of simple phenotypic tests for the specific identification of carbapenemase-producing strains, with varying degrees of endemicity observed based on the type of enzyme ( 12 ). Various methods have been developed for the detection of carbapenemases, including the Modified Hodge Test (MHT) ( 13 ), the Carba NP ( 14 ) test and its derivatives, the modified carbapenem inactivation method (mCIM) ( 15 ), as well as antibody-and PCR-based approaches ( 16 , 17 ). However, traditional forms of these methods lack specificity regarding the type of carbapenemase produced and cannot distinguish between Klebsiella pneumoniae carbapenemase (KPC) and metallo-beta-lactamase (MBL) carbapenemase. Despite the ability of antibody-and PCR-based methods to differentiate between KPC and MBL, they cannot discern carbapenem-resistant Enterobacteriaceae (CRE) from carbapenem-susceptible Enterobacteriaceae (CSE) isolates, particularly in cases where antimicrobial susceptibility is unknown. Furthermore, these methods are costlier and less accessible in local healthcare settings compared to traditional detection methods, thereby posing limitations in meeting clinical demands effectively. This study investigates the efficacy of the Disk Potentiation Test (DPT) for rapid and accurate identification of KPC and MBL carbapenemases, significant resistance determinants commonly found in Enterobacteriaceae isolates. By employing the DPT method, healthcare professionals can effectively identify KPC and MBL enzyme from CRE, aiding in the selection of appropriate antibiotics for patients with carbapenemase-producing CRE infections. This approach aims to mitigate the spread of carbapenemase-producing bacteria within healthcare settings. Materials & Methods Clinical isolates From January to June 2020, in a six-month period, a total of 147 gram-negative bacterial isolates were collected from various units of Evercare Hospital, previously called Apollo Hospital, Dhaka, were used for this study. During routine laboratory procedures, all showing resistance to Imipenem. Carbapenem non-susceptibility (Cns) was defined as a minimum inhibitory concentration (MIC) of ≥1 µg/ml for Enterobacteriaceae while Pseudomonas aeruginosa and Acinetobacter baumannii bacterial isolates Imipenem non-susceptibility was measured as an MIC of ≥2 µg/ml using the VITEK® 2 compact system. Following this, all Imipenem-resistant gram-negative bacteria were screened for KPC and MBL using the disc potentiation test (DPT) method ( 18 – 20 ). The isolates were obtained from outpatients (OPD), inpatients (IPD), and intensive care unit (ICU) patients at Evercare Hospital in Dhaka. The Microbiology & Immunology laboratory conducted phenotypic testing for KPC and MBL detection using the DPT method, which has demonstrated higher detection sensitivity in our previous reports ( 21 ). All experimental methods were carried out in accordance with guidelines and regulations approved by the “Research and Ethical Practice Committee” of Evercare Hospital, Dhaka, Bangladesh. Oral informed consent was obtained from the study subjects/patients or from their legal guardians and was approved by the “Research and Ethical Practice Committee” at Evercare Hospital, Dhaka, Bangladesh and the data were analyzed anonymously. Phenotypic confirmation of Disc potentiation test (DPT) for KPC detection The presence of KPC in K. pneumoniae and Enterobacteriaceae isolates was determined using boronic acid disk tests ( 18 ). A stock solution of phenylboronic acid (benzeneboronic acid) from Sigma-Aldrich, Steinheim, Germany, was prepared by dissolving 120 mg of phenylboronic acid in 3 ml of dimethyl sulfoxide. Then, 3 ml of sterile distilled water was added to this solution to reach a final concentration of 20 g/L. From this solution, 20 μl (containing 400 μg of phenylboronic acid) was dispensed onto commercially available Imipenem disks. Before application, the disks were dried and employed within 60 minutes ( 18 , 22 ). The test relies on comparing the zone diameters produced by discs containing Imipenem (IPM) with or without phenylboronic acid (PBA). A difference in zone diameter of >5 mm between the IPM and IPM + PBA discs was considered a positive result ( 22 – 23 ). The phenotypic confirmation of metallo-beta-lactamase (MBL) detection through DPT method The process involved the utilization of 0.5 M EDTA to inhibit MBL activity, as detailed by Yong et al. ( 20 ). This assay hinges upon comparing the zones generated by discs containing Imipenem (IPM) with or without 0.5 M EDTA Galani et al. ( 19 ) adapted the test by employing two 10 µg IPM discs. Subsequently, 10 µl of 0.5 M EDTA was applied to one of the IPM discs. The plates were incubated overnight at 37°C. In the DPT with 0.5 M EDTA, a growth inhibitory zone diameter enlargement exceeding 7 mm around the IPM + 0.5 M EDTA disc compared to IPM alone indicated a positive result for MBLs ( 19 ). Statistical Analysis For the statistical analyses, data were entered into an Excel spreadsheet and analyzed using the statistical software SPSS version 27.0 (IBM; Chicago, IL, USA) for Windows. Results Universal resistance to Imipenem was observed among all 147 bacterial isolates, particularly affecting individuals over 60 years, A total of 147 bacterial strains isolated from various clinical specimens were subjected to antibiogram test and exhibited 100% resistance to Carbapenems (Imipenem). We also investigated the distribution of resistant and susceptible isolates among different age groups. Results represented that the number of resistant isolates distribution was significantly high in >60 age group compare to other age groups ( Table 1 ). The percentage of bacterial isolates from male and female patient groups that showed 100% resistant to Imipenem were 78.2% and 21.8 % respectively ( Fig 1a ). The frequency of males and females affected by CRE in different unit of the hospital, the highest number of male patients affected carbapenem non-susceptible in ICU unit of the hospital ( Fig 1b ). The age-group “0 -15” and “>60” also represented the maximum resistant isolates from ICU hospital unit compare to IPD, OPD units ( Fig 1c ). In addition, the ICU unit displayed the highest prevalence of resistant isolates, with Acinetobacter baumannii being the most prominent, followed by Klebsiella pneumoniae , Pseudomonas aeruginosa , and Providencia stuartii , while Acinetobacter baumannii was most frequently found in ICU units, and Klebsiella pneumoniae , Pseudomonas aeruginosa , and Providencia stuartii were predominant in IPD units ( Fig 1d ). Download figure Open in new tab Fig 1. Resistance patterns of bacterial isolates across age and sex groups, depicting 100% resistance to carbapenems across all age categories, with the ">60" age group showing the highest number of resistant isolates. (a) Percentage of males and females affected by CRE. (b) Distribution of males and females affected by CRE in different hospital units. (c) Frequency of age-groups affected by CRE in different hospital units. (d) Frequency of clinically isolated carbapenem-resistant organisms (CRE) as etiological agents in clinical samples from various hospital units. View this table: View inline View popup Download powerpoint Table 1. Represents the antibiotic susceptibility profiles of various gram-negative strains (n = 147) across different age groups, all of which exhibited 100% resistance to Imipenem. The susceptibility profiles were assessed for Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia, Morganella morganii, Proteus mirabilis, Providencia stuartii, and Serratia marcescens (n = 147) using conventional culture and the VITEK®2 compact system. Minimum inhibitory concentration (MIC) analysis was conducted in accordance with CLSI guidelines. Additionally, the distribution of resistant and susceptible isolates among age groups was examined, revealing the highest number of resistant isolates in the age group ">60". The “>60” age group exhibited the highest resistance, while a majority of gram-negative isolates displayed resistance to commonly used antimicrobial agents at MIC range of ≥16 µg/ml. We also observed our observation of the distribution resistant bacterial isolates (n = 147) among age-groups data revealed that “>60” age-group showed the maximum number of resistant isolates ( Table 2 ). We also analyzed MIC (Imipenem) -wise distribution spectrum of all gram-negative isolates. Individual bacterial isolates (n = 147) from MIC range 1 - 15, 16 - >16 were treated against commonly used antimicrobial agents . Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Morganella morganii, Providencia stuartii, Burkholderia cepacia showed resistant to antimicrobial agents. Highest number of resistances was showed at MIC ≥16 µg/ml (n = 126) ( Table 2 ). View this table: View inline View popup Download powerpoint Table 2. Age-group distribution spectrum of various isolates and their MIC-based resistance level to Imipenem. A total of 147 bacterial isolates were subjected to antimicrobial susceptibility testing across different age groups, ranging from 0 to >60 years. Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia, Morganella morganii, Proteus mirabilis, Providencia stuartii, and Serratia marcescens exhibited resistance to commonly used antimicrobial agents, with isolates from various MIC ranges showing similar resistance patterns. Acinetobacter baumannii accounted for the highest detection of both KPC and MBL enzyme Our study found that Acinetobacter baumannii strains had the highest occurrence of both KPC and MBL enzymes. Among the bacterial isolates, 36 (24.49%) exhibited both KPC and MBL enzymes, 78 (53.06%) showed no enzyme presence, 3 (2.04%) had the KPC enzyme, and 30 (20.40%) had the MBL enzyme. Specifically, Acinetobacter baumannii accounted for the highest detection with 30 strains, followed by Klebsiella pneumoniae with 3 strains, Pseudomonas aeruginosa with 2 strains, and Escherichia coli with 1 strain ( Table 3 ). View this table: View inline View popup Download powerpoint Table 3. Crosstabulation analysis KPC and MBL enzymes profiles of various gram-negative strains (n=147). Interaction status of CRE strains to profile KPC and MBL drug-resistant enzymes among various gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia, Morganella morganii, Proteus mirabilis, Providencia stuartii, and Serratia marcescens were examined. Detection of KPC and MBL Enzymes in CRE Isolates via DPT Method Shows High Prevalence in ICU Patients We investigated the prevalence and distribution of KPC and MBL carbapenemases among clinical isolates. Among the clinical isolates investigated, 26.53 % (39 out of 147) were carriers of the KPC enzyme, and were predominantly identified in the ICU ( Fig 2a ). The distribution isolates are found to be KPC positive for these isolates varied across different units, with the highest prevalence noted in the ICU (23 out of 69), followed by IPD (14 out of 62) and OPD (2 out of 16). Notably, the majority of KPC-positive isolates exhibited resistance to carbapenems, with a significant portion falling within the MIC range, ≥16 µg/ml ( Fig 2b ). Acinetobacter baumannii emerged as the predominant bacterial strain producing the KPC enzyme accounting for 32 out of 61 isolates ( Fig 2c ). In addition, we analyzed clinical samples using the DPT method to detect CRE bacterial isolates with both KPC and MBL enzymes. The distribution and interaction of these drug-resistant enzymes were profiled among various gram-negative strains. The highest number of samples with both KPC and MBL enzymes were found in tracheal aspirates (n = 13), followed by suction tips (n = 4), blood (n = 4), wound swabs (n = 4), urine (n = 4), endotracheal tube tips (n = 3), sputum (n = 2), and pus (n = 2) ( Table 4 , Fig 2d ). On the other hand, among the 147 clinical isolates examined, 44.89% (66 out of 147) tested positive for MBL using the DPT test with 10 µl of 0.5 M EDTA, with the highest prevalence observed in the ICU (33 out of 69), followed by IPD (28 out of 62) and OPD (5 out of 16) ( Fig 2a ). The majority of MBL-positive isolates displayed carbapenem resistance, with the highest frequency observed ( Fig 2b ). Acinetobacter baumannii was identified as the predominant MBL-producing bacterial strain ( Fig 2c ). Additionally, the highest occurrences of bacterial isolates resistant to carbapenem carrying the MBL enzyme were found in tissue samples, followed by endotracheal tube tips, suction tips, blood, urine, tracheal aspirate, wound swab, pus, and sputum ( Fig 2d ). The majority of KPC and MBL both enzymes displayed ICU department n = 22, followed by IPD n = 12 and OPD n = 2 ( Table 5 ). Download figure Open in new tab Fig 2. Distribution and screening outcomes of KPC and MBL enzymes in clinically isolated carbapenem non-susceptible pathogens:: (a) The count of KPC and MBL enzyme screenings using various inhibitors across different hospital units, (b) The incidence of KPC and MBL enzyme isolation based on MIC distribution, (c) The number of KPC and MBL enzyme screenings affected by clinically isolated etiological agents using different inhibitors, and (d) The frequency of KPC and MBL enzyme screenings across various clinical samples. View this table: View inline View popup Download powerpoint Table 4. Crosstabulation analysis KPC and MBL enzymes profiles of various Clinical samples origin. In this analysis the distribution and interaction of clinical samples to profile KPC and MBL drug-resistant enzymes among various gram-negative strains, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia, Morganella morganii, Proteus mirabilis, Providencia stuartii, and Serratia marcescens were examined. View this table: View inline View popup Download powerpoint Table 5. Crosstabulation analysis KPC and MBL enzymes profiles of various unit of the hospital. In this analysis the distribution and interaction of ICU, IPD and OPD unit to profile KPC and MBL drug-resistant enzymes among various gram-negative strains, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia, Morganella morganii, Proteus mirabilis, Providencia stuartii, and Serratia marcescens were examined. Discussion Our study provides valuable insights into the prevalence and distribution of carbapenemases, particularly KPC and MBL, among clinical isolates, shedding light on critical factors influencing resistance patterns and patient outcomes. A striking finding of our investigation is the universal resistance to Imipenem observed among all 147 bacterial isolates, underscoring the urgent need for effective antimicrobial stewardship and infection control measures. Notably, individuals aged over 60 years exhibited a significantly higher prevalence of resistance, highlighting the potential impact of age-related factors on susceptibility to carbapenem-resistant infections. ( Table 1 ). The resistance levels to carbapenems, aminopenicillins, extended-spectrum cephalosporins, aminoglycosides, tetracyclines, and polypeptide antibiotics were unexpectedly high, consistent with findings from prior studies. For instance, Smita Sood in a study India Jaiypur has reported 88.33% of the isolates were found to be MBL producers, 4 (6.66%) were found to be MBL and KPC co-producers phenotypic confirmation was made by combined disc method ( 24 ), although the number of clinical isolates was insufficient to draw definitive conclusions including the age and sex distribution of the resistant isolates. Additionally, Datta et al. reported a significant increase in carbapenem resistance from 2.5% in 2002 to 52% in 2009, along with rising resistance to cefotaxime (75 to 97%) and piperacillin/tazobactam (55-84%) among Klebsiella pneumoniae strains isolated from bloodstream infections in Delhi ( 25 ). Likewise, research conducted in southern India reported the rates of carbapenem resistance among hospital-acquired Klebsiella pneumoniae isolates: Meropenem 43.6%, Imipenem 32%, Ertapenem 20.3%, and Colistin 60% ( 26 ). More recently, a study conducted in Dhaka, Bangladesh, reported a 4.8% incidence of resistant against carbapenems (Meropenem and Imipenem) and all the carbapenem resistant isolates were found to be KPC positive however were detected using the modified Hodge test methods, typically requires more steps and interpretation, making it relatively more complex which has less accessibility in local hospital settings ( 27 ). This pervasive resistance underscores the urgent need for effective antimicrobial stewardship programs and infection control measures. Gender disparities in antimicrobial resistance were observed, with a higher percentage of resistant isolates identified among male patients compared to females, especially in the ICU, highlighting the need for targeted surveillance. The ICU exhibited a significant concentration of carbapenem-resistant strains compared to OPD and IPD ( Fig 1 ), particularly among patients over 60 years old, with Klebsiella pneumoniae was the most prevalent organism isolated followed by Acinetobacter baumannii , Pseudomonas aeruginosa , and others ( Table 2 ). We also investigated the prevalence and distribution of KPC and MBL carbapenemases among clinical isolates. The finding revealed that 26.5% of the isolates carried the KPC enzyme, with a higher prevalence in the ICU. A high-level resistance across gram-negative isolates were observed at MIC values above 16 µg/ml for Imipenem, with Acinetobacter baumannii emerged as the primary producer of the KPC enzyme. Additionally, 45% of the clinical isolates tested positive for MBL, predominantly in the ICU. Isolates resistant to carbapenems due to KPC and MBL enzyme exhibited distinct frequencies among clinical samples, with tissue samples, endotracheal tube tips, suction tips, blood, urine, tracheal aspirates, wound swabs, pus, and sputum specimens demonstrating varying levels of resistance ( Fig 2 ). In our investigation of carbapenemase-producing isolates by specimen types, predominantly observed in tracheal aspirate followed by urine, blood, wound swab, and others. A dissimilarity in the trend was observed by a study in Bangalore, India, where most carbapenem-resistant Klebsiella pneumoniae isolates were obtained from urine samples ( 28 ). And, evidenced by studies conducted in the USA and India have reported a higher prevalence of Klebsiella pneumoniae , Carbapenem-resistant (CRKP) in respiratory or blood specimens from adult male patients admitted to the ICU ( 29 ). Our study identified Acinetobacter baumannii as the predominant carrier of both KPC and MBL enzymes among the bacterial isolates and was accounted for the highest detection of both the enzymes, with 30 strains, followed by Klebsiella pneumoniae , Pseudomonas aeruginosa , and Escherichia coli ( Table 3 ). Furthermore, our analysis using the Disc Potentiation Test (DPT) method revealed a significant prevalence of KPC and MBL enzymes in clinical samples. The distribution showed that tracheal aspirates had the highest number of samples with both enzymes, followed by suction tips, blood, wound swabs, urine, endotracheal tube tips, sputum, and pus ( Table 4 ). These findings highlight the urgent need for robust monitoring and control measures to manage the spread of multidrug-resistant organisms, particularly Acinetobacter baumannii , in healthcare settings. The high prevalence of KPC and MBL enzymes in tracheal aspirates suggests a significant infection and transmission risk, underscoring the importance of strict infection control practices and the development of novel therapeutic strategies. Effective detection and management strategies of these resistant strains is critical to mitigating their impact on public health. Data Availability All data generated or analyzed during this study are included in this published article Declaration statement Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that there are no conflicts of interest. Acknowledgments This work was partially supported by personal fund of Dr. NA in Evercare Hospital Dhaka, Bangladesh. All authors contributed to this research work from their responsibility to the society. We are thankful to our colleagues, and laboratory attendant Ilias Hossain who supported this research work. We are grateful to the patients for providing samples and for their participation in our research. References 1. ↵ Rhomberg PR , Jones RN . Summary trends for the meropenem yearly susceptibility test information collection program: a 10-year experience in the United States (1999–2008) . Diagnostic microbiology and infectious disease . 2009 Dec 1; 65 ( 4 ): 414 – 26 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Queenan AM , Bush K . Carbapenemases: the versatile β-lactamases . Clinical microbiology reviews . 2007 Jul; 20 ( 3 ): 440 – 58 . OpenUrl Abstract / FREE Full Text 3. ↵ Paterson DL , Bonomo RA . Extended-spectrum β-lactamases: a clinical update . Clinical microbiology reviews . 2005 Oct; 18 ( 4 ): 657 – 86 . OpenUrl Abstract / FREE Full Text 4. ↵ Livermore DM , Woodford N . The β-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter . TRENDS in Microbiology . 2006 Sep 1; 14 ( 9 ): 413 – 20 . OpenUrl CrossRef PubMed Web of Science 5. ↵ Hrabák J , Chudáčková E , Papagiannitsis CC . Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories . Clinical Microbiology and Infection . 2014 Sep 1; 20 ( 9 ): 839 – 53 . OpenUrl CrossRef PubMed 6. ↵ Kumarasamy KK , Toleman MA , Walsh TR , Bagaria J , Butt F , Balakrishnan R , Chaudhary U , Doumith M , Giske CG , Irfan S , Krishnan P . Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study . The Lancet infectious diseases . 2010 Sep 1; 10 ( 9 ): 597 – 602 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Tsakris A , Poulou A , Pournaras S , Voulgari E , Vrioni G , Themeli-Digalaki K , Petropoulou D , Sofianou D . A simple phenotypic method for the differentiation of metallo-β-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates . Journal of antimicrobial chemotherapy . 2010 Aug 1; 65 ( 8 ): 1664 – 71 . OpenUrl CrossRef PubMed Web of Science 8. Naas T , Cuzon G , Villegas MV , Lartigue MF , Quinn JP , Nordmann P . Genetic structures at the origin of acquisition of the β-lactamase bla KPC gene . Antimicrobial agents and chemotherapy . 2008 Apr; 52 ( 4 ): 1257 – 63 . OpenUrl Abstract / FREE Full Text 9. ↵ Ikonomidis A , Tokatlidou D , Kristo I , Sofianou D , Tsakris A , Mantzana P , Pournaras S , Maniatis AN . Outbreaks in distinct regions due to a single Klebsiella pneumoniae clone carrying a bla VIM-1 metallo-β-lactamase gene . Journal of clinical microbiology . 2005 Oct; 43 ( 10 ): 5344 – 7 . OpenUrl Abstract / FREE Full Text 10. ↵ Picão RC , Cardoso JP , Campana EH , Nicoletti AG , Petrolini FV , Assis DM , Juliano L , Gales AC . The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage . Diagnostic microbiology and infectious disease . 2013 May 1; 76 ( 1 ): 80 – 5 . OpenUrl CrossRef PubMed 11. ↵ Goic-Barisic I , Hrenovic J , Kovacic A , Musić MŠ . Emergence of oxacillinases in environmental carbapenem-resistant Acinetobacter baumannii associated with clinical isolates . Microbial drug resistance . 2016 Oct 1; 22 ( 7 ): 559 – 63 . OpenUrl PubMed 12. ↵ Farzana R , Shamsuzzaman SM , Mamun KZ . Isolation and molecular characterization of New Delhi metallo-beta-lactamase-1 producing superbug in Bangladesh . The Journal of infection in developing countries . 2013 Mar 14; 7 ( 03 ): 161 – 8 . OpenUrl PubMed 13. ↵ Carvalhaes CG , Picao RC , Nicoletti AG , Xavier DE , Gales AC . Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results . Journal of antimicrobial chemotherapy . 2010 Feb 1; 65 ( 2 ): 249 – 51 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Laolerd W , Akeda Y , Preeyanon L , Ratthawongjirakul P , Santanirand P . Carbapenemase-producing carbapenem-resistant Enterobacteriaceae from Bangkok, Thailand, and their detection by the Carba NP and modified carbapenem inactivation method tests . Microbial Drug Resistance . 2018 Sep 1; 24 ( 7 ): 1006 – 11 . OpenUrl PubMed 15. ↵ Butler-Wu SM , Abbott AN . Is this the carbapenemase test we’ve been waiting for? A multicenter evaluation of the modified carbapenem inactivation method . Journal of Clinical Microbiology . 2017 Aug; 55 ( 8 ): 2309 – 12 . OpenUrl Abstract / FREE Full Text 16. ↵ Kieffer N , Poirel L , Nordmann P . Rapid immunochromatography-based detection of carbapenemase producers . Infection . 2019 Aug 1; 47 : 673 – 5 . OpenUrl CrossRef PubMed 17. ↵ Cointe A , Walewski V , Hobson CA , Doit C , Bidet P , Dortet L , Bonacorsi S , Birgy A . Rapid carbapenemase detection with Xpert Carba-R V2 directly on positive blood vials . Infection and Drug Resistance . 2019 Oct 23: 3311 – 6 . 18. ↵ Girgis SA , Othman HB , Kassem NN , Abdou SM . Evaluation of boronic acid disk for detection of Klebsiella pneumoniae carbapenemase in Klebsiella pneumoniae . Int J Curr Microbiol App Sci . 2015 Jun 10; 4 ( 5 ): 772 – 83 . OpenUrl 19. ↵ Galani I , Rekatsina PD , Hatzaki D , Plachouras D , Souli M , Giamarellou H . Evaluation of different laboratory tests for the detection of metallo-β-lactamase production in Enterobacteriaceae . Journal of antimicrobial chemotherapy . 2008 Mar 1; 61 ( 3 ): 548 – 53 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Yong D , Lee K , Yum JH , Shin HB , Rossolini GM , Chong Y . Imipenem-EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp . Journal of clinical microbiology . 2002 Oct; 40 ( 10 ): 3798 – 801 . OpenUrl Abstract / FREE Full Text 21. ↵ Das SK , Sheikh A , Ara N , Biswas SM , Chowdhury A , Zahra FA , Keya CA . Evaluation of Disk Potentiation Test (DPT) and Double Disk Synergy Test (DDST) for The Detection of Metallo-β-lactamases (MBLs) in Clinical Isolates of Bangladesh . medRxiv . 2022 Dec 27: 2022 – 12 . 22. ↵ Tsakris A , Kristo I , Poulou A , Themeli-Digalaki K , Ikonomidis A , Petropoulou D , Pournaras S , Sofianou D . Evaluation of boronic acid disk tests for differentiating KPC-possessing Klebsiella pneumoniae isolates in the clinical laboratory . Journal of clinical microbiology . 2009 Feb; 47 ( 2 ): 362 – 7 . OpenUrl Abstract / FREE Full Text 23. ↵ Kuinkel S , Acharya J , Dhungel B , Adhikari S , Adhikari N , Shrestha UT , Banjara MR , Rijal KR , Ghimire P . Biofilm formation and phenotypic detection of ESBL, MBL, KPC and AmpC enzymes and their coexistence in Klebsiella spp. isolated at the National Reference Laboratory, Kathmandu, Nepal . Microbiology Research . 2021 Aug 18; 12 ( 3 ): 49 . OpenUrl 24. ↵ Sood S . Identification and differentiation of carbapenemases in Klebsiella pneumoniae: a phenotypic test evaluation study from Jaipur , India. Journal of clinical and diagnostic research: JCDR . 2014 Jul; 8 ( 7 ): DC01 . OpenUrl 25. ↵ Datta S , Wattal C , Goel N , Oberoi JK , Raveendran R , Prasad K . A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli & Klebsiella pneumoniae in a tertiary care hospital . Indian Journal of Medical Research . 2012 Jun 1; 135 ( 6 ): 907 – 12 . OpenUrl PubMed 26. ↵ Parveen RM , Harish BN , Parija SC . Emerging carbapenem resistance among nosocomial isolates of Klebsiella pneumoniae in South India . Int J Pharma Bio Sci . 2010 ; 1 ( 2 ): 1 – 1 . OpenUrl 27. ↵ Hayder N , Hasan Z , Afrin S , Noor R . Determination of the frequency of carbapenemase producing Klebsiella pneumoniae isolates in Dhaka city , Bangladesh. Stamford Journal of Microbiology . 2012 ; 2 ( 1 ): 28 – 30 . OpenUrl 28. ↵ Nagaraj S , Chandran SP , Shamanna P , Macaden R . Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in south India . Indian Journal of Medical Microbiology . 2012 Jan 1; 30 ( 1 ): 93 – 5 . OpenUrl PubMed 29. ↵ Braykov NP , Eber MR , Klein EY , Morgan DJ , Laxminarayan R. Trends in resistance to carbapenems and third-generation cephalosporins among clinical isolates of Klebsiella pneumoniae in the United States, 1999–2010 . Infection Control & Hospital Epidemiology . 2013 Mar; 34 ( 3 ): 259 – 68 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted January 03, 2025. Download PDF Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method Sumon Kumar Das , Nikhat Ara , Suma Mita Biswas , Afzal Sheikh medRxiv 2024.12.30.24319797; doi: https://doi.org/10.1101/2024.12.30.24319797 Share This Article: Copy Citation Tools Prevalance of Carbapenemase Production Among Clinical Gram-Negative Isolates in Bangladesh: A Comprehensive Study Using Disc Potentiation Test (DPT) Method Sumon Kumar Das , Nikhat Ara , Suma Mita Biswas , Afzal Sheikh medRxiv 2024.12.30.24319797; doi: https://doi.org/10.1101/2024.12.30.24319797 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Infectious Diseases (except HIV/AIDS) Subject Areas All Articles Addiction Medicine (568) Allergy and Immunology (863) Anesthesia (297) Cardiovascular Medicine (4421) Dentistry and Oral Medicine (443) Dermatology (382) Emergency Medicine (606) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1507) Epidemiology (15212) Forensic Medicine (30) Gastroenterology (1121) Genetic and Genomic Medicine (6581) Geriatric Medicine (667) Health Economics (996) Health Informatics (4520) Health Policy (1366) Health Systems and Quality Improvement (1611) Hematology (539) HIV/AIDS (1264) Infectious Diseases (except HIV/AIDS) (15906) Intensive Care and Critical Care Medicine (1103) Medical Education (620) Medical Ethics (144) Nephrology (667) Neurology (6580) Nursing (345) Nutrition (998) Obstetrics and Gynecology (1141) Occupational and Environmental Health (956) Oncology (3324) Ophthalmology (970) Orthopedics (369) Otolaryngology (420) Pain Medicine (435) Palliative Medicine (129) Pathology (663) Pediatrics (1689) Pharmacology and Therapeutics (691) Primary Care Research (710) Psychiatry and Clinical Psychology (5433) Public and Global Health (9212) Radiology and Imaging (2193) Rehabilitation Medicine and Physical Therapy (1368) Respiratory Medicine (1194) Rheumatology (593) Sexual and Reproductive Health (709) Sports Medicine (529) Surgery (709) Toxicology (99) Transplantation (288) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ff59cedbb15df88',t:'MTc3OTM4NzgxNA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();