Carbapenem-resistant Clinical Isolates Research Articles

Biofilm Formation and Antibiotic Resistance Profiles in Carbapenemase-Producing Gram-Negative Rods—A Comparative Analysis between Screening and Pathological Isolates

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.

Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli

BackgroundAztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches.Methods96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher).ResultsAccording to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%).ConclusionsThe ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4μg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 μg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to β-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: β-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including β-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

In vitro activity of sulbactam-durlobactam against colistin-resistant and/or cefiderocol-non-susceptible, carbapenem-resistant Acinetobacter baumannii collected in U.S. hospitals.

The activity of a novel β-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC50/MIC90 values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

Colistin and Polymyxin B sensitivity by broth microdilution among carbapenem-resistant clinical isolates of Gram-negative bacilli

Introduction and Aim: The increasing incidence of multidrug-resistant gram-negative bacilli (MDR-GNB) in recent times has led to the revival of older neglected antibiotics such as Polymyxins. Hence, there is a need to determine the minimum inhibitory concentration (MIC) of these antibiotics by conventional and automated antibiotic sensitivity testing methods to assess their clinical utility. This study aimed to assess the in vitro sensitivity of clinical isolates grown with carbapenem-resistant GNB (CR-GNB) to Colistin and Polymyxin B by broth micro-dilution method (BMD). Materials and Methods: Thirty isolates of CR-GNB obtained from various clinical samples received in the microbiology laboratory of Yenepoya Medical College Hospital between August 2018 to November 2019 were included in the study. In vitro assessment of Colistin and Polymyxin B sensitivity among CR-GNB was performed by BMD as per CLSI guidelines. Results: Twelve isolates (40%) were carbapenemase producers. Among the 30 isolates of CR-GNB, 56.6% and 63.3 % of them were sensitive to Colistin and Polymyxin, respectively. Colistin MICs of 12 isolates obtained by BMD assay were concordant with the MICs obtained by BD Phoenix. Conclusion: The results of this study indicate that Colistin and Polymyxin B can be considered as reserve therapeutic agents for treating infections caused by CR-GNB after obtaining the in vitro sensitivity results.

Genome analysis of triple phages that curtails MDR E. coli with ML based host receptor prediction and its evaluation

Infections by multidrug resistant bacteria (MDR) are becoming increasingly difficult to treat and alternative approaches like phage therapy, which is unhindered by drug resistance, are urgently needed to tackle MDR bacterial infections. During phage therapy phage cocktails targeting different receptors are likely to be more effective than monophages. In the present study, phages targeting carbapenem resistant clinical isolate of E. coli U1007 was isolated from Ganges River (U1G), Cooum River (CR) and Hospital waste water (M). Capsid architecture discerned using TEM identified the phage families as Podoviridae for U1G, Myoviridae for CR and Siphoviridae for M phage. Genome sequencing showed the phage genomes varied in size U1G (73,275 bp) CR (45,236 bp) and M (45,294 bp). All three genomes lacked genes encoding tRNA sequence, antibiotic resistant or virulent genes. A machine learning (ML) based multi-class classification model using Random Forest, Logistic Regression, and Decision Tree were employed to predict the host receptor targeted by receptor binding protein of all 3 phages and the best performing algorithm Random Forest predicted LPS O antigen, LamB or OmpC for U1G; FhuA, OmpC for CR phage; and FhuA, LamB, TonB or OmpF for the M phage. OmpC was validated as receptor for U1G by physiological experiments. In vivo intramuscular infection study in zebrafish showed that cocktail of dual phages (U1G + M) along with colsitin resulted in a significant 3.5 log decline in cell counts. Our study highlights the potential of ML tool to predict host receptor and proves the utility of phage cocktail to restrict E. coli U1007 in vivo.

Assessment of in vitro colistin susceptibility of carbapenem-resistant clinical Gram-negative bacterial isolates using four commercially available systems & Whole-genome sequencing: A diagnostic accuracy study

AimTo analyze the diagnostic utility of commercially available platforms and Whole-genome sequencing (WGS) for accurate determination of colistin susceptibility test results. Material & methodsAn exploratory diagnostic accuracy study was conducted in which sixty carbapenem-resistant Gram-negative bacteria were subjected to identification and AST using MALDI-TOF MS & MicroScan walkaway 96 Plus. Additional AST was performed using the BD Phoenix system and Mikrolatest colistin kit. The test isolates were subjected to Vitek-2 and WGS at CRL, Bengaluru. ResultsThere was no statistically significant agreement between the colistin susceptibility results obtained by WGS, with those of commercial phenotypic platforms. The MicroScan 96 Plus had the highest sensitivity (31 %) & NPV (77 %), and the BD Phoenix system had the highest specificity (97 %) and PPV (50 %), respectively, for determining colistin resistance. ConclusionThe utility of WGS as a tool in AMR surveillance and validation of phenotypic AST methods should be explored further.

713. Prevalence of Microbicide Resistance Genes in Clinical Isolates of Acinetobacter baumannii

Abstract Background The persistence of Acinetobacter on different surfaces and the continuous exposure of bacteria to disinfectants such as quaternary ammonium compounds (QACs) in the healthcare environment have led to microbicide tolerance. Acinetobacter baumannii have also rapidly acquired carbapenem resistance. Here we determined the prevalence of microbicide resistance genes in carbapenem susceptible and resistant clinical isolates of Acinetobacter. Methods All clinical isolates were collected from two separate hospitals in Detroit between 2017-2021. Following DNA isolation using Qiagen kit, Nextera Flex kit was used for library preparation of Acinetobacter clinical isolates. Whole genome sequencing (WGS) was performed via Illumina NextSeq 550 using the prepared DNA library. The FASTQ files from the sequencing run were then subjected to bioinformatic analysis with Bionumerics software v7.6. ResFinder was used for determination of presence or absence of QAC resistant genes and results were tabulated by Carbapenem-Resistant Acinetobacter baumannii (CRAb) or Carbapenem-Susceptible Acinetobacter baumannii (CSAb) with or without the QAC resistant genes. Results Out of the 139 Acinetobacter patient isolates, 70/139 (50.4%) were CRAb containing carbapenemase blaOXA-23 and 69/139 (49.6%) belonged to CSAb (Table 1). Figure 1. shows the prevalence of antimicrobial resistance (AMR) genes in CRAb and CSAb isolates with qacE genes. For CRAb isolates, 56% had qacE genes while 39% of CSAb had qacE genes. About 42% of CRAb isolates were multi-drug resistant (MDR), indicating a prevalence of qacE genes among the MDR isolates. Table 2 shows the presence of qacE genes by sequence type (ST). In the ST2 (Pasteur) and ST406 (Pasteur) strains, qacE gene was mainly present. Interestingly, most of the Acinetobacter isolates that belong to ST2 (ST195 and ST281, Oxford) are CRAb with qacE gene.Figure 1:Different AMR genes and qacE in both CRAb and CSAb isolates Conclusion Clinical isolates in our study had an even distribution of both CRAb and CSAb. The presence of qacE gene was more prevalent in the CRAb isolates compared to the CSAb isolates. While different STs harbored the qacE gene, the prevalence of qacE gene was highest in ST2 among all isolates. Future studies are needed to determine the reduced susceptibility to other commonly used hospital disinfectants. Disclosures Keith S. Kaye, MD, MPH, Abbvie: Advisor/Consultant|Abbvie: Honoraria|Entasis: Advisor/Consultant|Entasis: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|Merck: Advisor/Consultant|Merck: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|VenatoRx: Advisor/Consultant|VenatoRx: Honoraria

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid

Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.
 Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.
 Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.
 Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.

Colistin Susceptibility Testing by Colistin Broth Disk Elution MIC Method among Carbapenem-resistant Gram-negative Blood Culture Clinical Isolates in a Tertiary Care Setting, East Delhi, India.

Antibiotic resistance is a growing concern for bloodstream infections (BSIs), especially with the emergence of multidrug-resistant (MDR) gram-negative bacteria. In this study, we aimed to assess the pattern of colistin susceptibility using the colistin broth disc elution (CBDE) method among carbapenem-resistant gram-negative clinical isolates from blood cultures in a high burden tertiary healthcare setting in East Delhi. A total of 106 carbapenem-resistant gram-negative clinical isolates were tested. The most common isolates were Klebsiella pneumoniae, Escherichia coli, Enterobacter species, and Klebsiella oxytoca by CBDE method. All the carbapenem resistant gram-negative bacterial blood culture isolates showed intermediate colistin susceptibility. This was statistically significant by chi-square test (p<0.5). This study highlights the need to monitor colistin resistance trends in the face of increasing antimicrobial resistance. Accurate surveillance of emerging colistin resistance is crucial for effective management of BSIs caused by carbapenem-resistant gram-negative bacteria.

In vitro activity of cefiderocol against carbapenem-resistant Acinetobacter baumannii clinical isolates: a single center experience.

Introduction: Cefiderocol (CFDC) is a novel siderophore cephalosporin with broad-spectrum activity against carbapenem-resistant Gram-negative bacteria. Gram-negative bacterial isolates' resistance to cefiderocol (CFDC) is emerging in clinical practice.

Molecular Detection of Carbapenem Resistance in Clinical Isolates of Klebsiella pneumoniae in Tertiary Care Hospital

Antibiotic resistance has become a serious global threat, mainly due to misuse, overuse of antibiotics and non-compliance with infection control protocol. Superbugs are multidrug-resistant (MDR) and extended drug-resistant (XDR) bacteria, mainly Klebsiella pneumoniae and Escherichia coli from the Enterobacteriaceae family, which cause opportunistic infections and raise death rates and hospital expenditures. The present study was conducted at a tertiary care teaching hospital to study the epidemiology and molecular detection of carbapenem-resistant K. pneumoniae isolated from various clinical specimens. 240 K. pneumoniae isolates were collected from January 2020 to December 2021 at the Bacteriology laboratory, Index Medical College and Hospital, Indore. All isolates were analyzed for carbapenem resistance by the conventional disc diffusion method. All carbapenem-resistant isolates were tested for carbapenemase production using the phenotypic double-disk synergy test (DDST) and modified Hodge test (MHT) as per 2020 CLSI guidelines. All isolates were negative by phenotypic methods, further confirmed by conventional PCR to detect the gene responsible for carbapenemase production. 240 isolates of K. pneumoniae were included during the study periods. Out of 240 isolates, 102 isolates were found resistant to carbapenem drugs. All 102 isolates were confirmed carbapenemase and MBL producers by MHT and DDST tests. Among 102, 60 isolates were found to be MBL producers negative by MHT and DDST tests. Sixty phenotypic negative carbapenem-resistant isolates were tested by conventional PCR. One or more carbapenemase genes were detected in 61.0% of isolates. The blaKPC was detected in 13/60 (21%) isolates, followed by blaNDM 10/60 (16%) isolates, followed by blaVIM in 6/60(10%), blaOXA-48 in 5/60 (8%) and blaIMP in 3/60(5%) isolates. K. pneumoniae produces carbapenemase, which enhances resistance to the carbapenem class of antibiotics. The simultaneous detection of these resistance genes expressed by Klebsiella pneumoniae might be managed by early detection and adhering to antibiotic policies that limit the use of antibiotics.

Prevalence and Phenotypic Detection of Carbapenem-Resistant Enterobacter Species from the Clinical Specimens of a Tertiary Care Hospital in Bangladesh

Background: Carbapenem-resistant Enterobacter (CRE) is an emerging threat spread rapidly around the world in the past few years, posing substantial hazards to human health. Objective: The study was conducted to evaluate the prevalence of carbapenem resistance and to detect carbapenemase producers phenotypically from patients of a major tertiary care hospital, in Bangladesh. Methods: Prospective cross-sectional study was conducted from July 2018 to June 2019, at the Dhaka Medical College hospital. Patients admitted to different wards, and intensive care units, visited the outpatient department, and samples received in the microbiology department were included in this study. A total of 350 clinical samples were collected, inoculated, and incubated in accordance to the standard protocol. Identification was done using the standard biochemical method. Antimicrobial susceptibility testing of commonly used antibiotics including imipenem was done using Kirby Bauer disk diffusion method. All the clinical isolates of Enterobacter were screened for carbapenem resistance as per CLSI guidelines. Such strains were then subjected to phenotypic confirmation of carbapenemase production by the Modified Hodge test. All isolates that gave a positive screening test were further evaluated for Metallo-β lactamase (MBL) production. MBL was further detected by Combined Disc Test (CD) using a combination of Imipenem and Imipenem-EDTA and double disc synergy (DDS) test. Result: A total of 350 clinical specimens were analyzed, of which 224 (65.14%) isolates yielded growth. Among them, 28 (12.28%) Enterobacter were isolated of which 12 (42.86%) were found to be Imipenem resistant (14.25%) and were labeled 'Carbapenem-resistant Enterobacter" or CRE. Minimum inhibitory concentration (MIC) was determined among the carbapenem-resistant clinical isolates. Modified Hodge test (MHT) performed on the 12 carbapenem-resistant isolates showed 9 (75%) isolates to be carbapenemase enzyme producers. Nine out of twelve carbapenem-resistant isolates are Metallo-beta-lactamase producers detected by combined disc test (CDT) and eight (66.67%) MBL producers detected by DDS test. Conclusion: The finding from this study revealed a high prevalence of carbapenem resistance among isolated Enterobacter species in hospital settings as well as community levels. As there are limited treatment regimens, therefore, the timely and accurate detection of carbapenem-resistant Enterobacter species is essential for the clinical treatment and prevention of infections. Simple phenotypic methods like MHT can be used for the rapid detection of carbapenemases, and these are expected to be used routinely in clinical microbiology laboratories. Update Dent. Coll. j: 2023; 13(1): 17-22

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe

ObjectivesTo characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. MethodsA. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. ResultsIsolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. ConclusionColistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Carbapenem-resistant Acinetobacter baumannii healthcare-associated infections: antimicrobial resistance and its spread as a global threat.

Carbapenem-resistant Acinetobacter baumannii healthcare-associated infections: antimicrobial resistance and its spread as a global threat.

Meropenem Incorporated ZnO Nanoflakes as Nano Antibiotics: Efficient Antimicrobial Activity against Metallo β-lactamase Producing Clinical Isolates

The number of fatalities caused by multidrug-resistant (MDR) bacteria is over 700,000 annually due to widespread antibiotic usage. So, there is a need of new antibiotics, materials that work like antibiotics, or combinations of antibiotics with nanomaterials that could help in treating the infections which is caused by MDR bacteria. The present study describes the synthesis of ZnO nanoflakes using a co-precipitation method. The ZnO nanoflakes and ZnO nanoflakes combinations with carbapenem antibiotics were tested against carbapenem-resistant (CR) clinical isolates. The SEM analysis showed surface morphology of the synthesized nanoflakes-like structure of ZnO. All 67 CR isolates were tested and showed inhibitory action at varying concentrations of ZnO nanoflakes. ZnO nanoflakes were found to have an inhibitory effect against Escherichia coli and Klebsiella pneumoniae at lowest concentration of 1.25 mg.ml-1 of ZnO NPs with average zone size (mean ±SD) 1.91±2.94 mm and 2.00±4.14 mm and the average zone size of ZnO nanoflakes against Acinetobacter baumanni and Pseudomonas aeruginosa was 9.89±0.76 mm and 10.17±0.39 mm at 2.5 mg.ml-1 concentration. The combined action of ZnO nanoflakes with Meropenem 10 mcg demonstrated synergetic activity against CR pathogens, with an average zone of inhibition measuring 15.2 mm in diameter. ZnO nanoflakes illustrated considerable antibacterial activity against MBL-producing gram-negative clinical isolates at the lowest concentration. Chemically synthesized ZnO nanoflakes may offer a superior future expectation as a nano-antibiotic to treat the infection caused by CRE bacteria.

Carbapenem resistance in Acinetobacter pittii isolates mediated by metallo-β-lactamases.

To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described. These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes.

First report of Molecular Epidemiology of Carbapenem Resistant Enterobacteriaceae from a Tertiary Level Hospital in Rajasthan, Western India

Background: Carbapenem Resistant Enterobacteriaceae (CRE) are emerging at an alarming rate and pose a significant global threat. Objective: To conduct phenotypic and genotypic characterization of CRE strains from Rajasthan, Western India. Methodology: This was a prospective observational study conducted in Department of Microbiology, Dr S.N. Medical College, Jodhpur, Rajasthan from October to December 2018. All clinical samples received during the study period were processed and bacterial identification and antimicrobial susceptibility tests were performed according to standard microbiological guidelines. A total of 14 non duplicate carbapenem resistant clinical isolates of E coli and K pneumoniae were included in the study and subjected to Rapidec Carba NP test. Carbapenemase‑ encoding genes were amplified by multiplex polymerase chain reaction (PCR). PCR amplified products from three random isolates were subjected to Sanger sequencing. Results: Amikacin remained active against 36% isolates. All isolates were found to be susceptible to colistin and tigecycline. Carbapenemase production by Rapidec Carba NP test was noted in all (14/14) study isolates. All isolates were found to harbour ≥ 1 carbapenemase gene. The most common resistance gene observed was blaoxa (86%) followed by blaNDM (79%). None of the CRE isolates included in our study showed production of KPC enzymes. The sequences were analysed using BLAST analysis and were confirmed to be matching to OXA-48/181 and NDM-1. Conclusions: Growing carbapenem resistance is an important issue which needs urgent attention and blaOXA is an emerging mechanism of resistance among clinical CRE isolates in our setting Keywords: Carbapenem resistant Enterobacteriaceae, Phenotypic tests, Carbapenemase gene, Polymerase chain reaction