Imipenem Resistance Research Articles

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.
 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.
 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.
 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.

Molecular study of blaVIM and blaIMP genes in Acinetobacter baumannii strains isolated from burn patients in Duhok City, Iraq.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogenic bacterium mainly associated with hospital acquired infections and in immunocompromised individuals who stay in hospitals for a long time. In recent years, it has become increasingly resistant to many different types of antibiotics. The production of the metallo-beta-lactamase (MBL) enzyme is one of the primary causes of this resistance. This study aimed to detect the presence of MBL genes that belong to the verona integrin metallo-β-lactamase (bla-VIM) and imipenemase (bla-IMP) groups in the isolates of Acinetobacter baumannii from burn patients. One hundred and seventeen (117) isolates of A. baumannii were obtained from patient specimens using traditional methods followed by using the VITEK 2 (BioMérieux, Les Pennes-Mirabeau, France) identification system. Metallo β-lactamases were detected in the imipenem-resistant strains by using imipenem disks on Muller-Hinton agar. The polymerase chain reaction (PCR) technique was utilized to examine 117 isolates for the detection of MBLs encoding genes such as bla-VIM, and bla-IMP. Imipenem resistance was detected in 78.6% of the patients. The PCR assays of the isolates identified bla-VIM-1, bla-VIM-2, bla-IMP-1 and bla-IMP-2 genes at the rates of 17%, 40.1%, 29.9% and 4.2%, respectively. The findings suggest that the majority of A. baumannii isolates harbour one or more of the detected genes, signifying that the production of MBLs plays a pivotal role in resistance mechanisms.

Occurrence of Imipenem-Resistant Uropathogenic Escherichia coli in Pregnant Women: An Insight into Their Virulence Profile and Clonal Structure.

Uropathogenic Escherichia coli (UPEC) is the predominant pathogen in Urinary Tract Infection (UTI) in pregnant and non-pregnant women. Limited studies were initiated to explore UPEC from pregnant women with respect to imipenem resistance, pathogenicity, and their clonal lineage. In this study, imipenem resistance, phylogenetic background, virulence-associated genes, and clonal characteristics in UPECs isolated from pregnant and non-pregnant cohorts were investigated. E. coli was identified biochemically from urine culture-positive samples from pregnant and non-pregnant women. Carbapenem (meropenem, ertapenem, imipenem) susceptibility was determined by Kirby-Bauer disk diffusion test. The pathogenic determinants were identified by PCR. MEGA 11 was used to interpret clonal lineages from MLST. GraphPad Prism 8.0 and SPSS 26.0 were used for statistical interpretation. Results indicated highest resistance against imipenem compared to meropenem and ertapenem in UPECs isolated from pregnant (UPECp; 63.89%) and non-pregnant (UPECnp; 87.88%) women. Although phylogroup E was predominant in both imipenem-resistant isolates, acquisition of virulence factors was higher among UPECnp than UPECp. Akin to this observation, the presence of PAI III536 and PAI IV536 was statistically significant (p < 0.05) in the former. MLST analysis revealed similar clonal lineages between UPECnp and UPECp, which showed an overall occurrence of ST405 followed by ST101, ST410, ST131, and ST1195 in UPECnp and ST167 in UPECp, respectively, with frequent occurrence of CC131, CC405. Therefore, imipenem-resistant UPECp although discrete with respect to their virulence determinants when compared to UPECnp shared similar STs and CCs, which implied common evolutionary history. Thus, empiric treatment must be restricted in UTIs to especially protect maternal and fetal health.

Imipenem heteroresistance but not tolerance in Haemophilus influenzae during chronic lung infection associated with chronic obstructive pulmonary disease.

Antibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients.

Analysis of blood culture data at a university hospital: bacterial distribution and cumulative antimicrobial resistance (2016–2020)

Background: The distribution of bacteria isolated from bloodstream infections and cumulative antimicrobial susceptibility data are the basis for empirical decisions regarding antibiotics as an initial treatment. Therefore, it is important to consistently collect blood culture results of individual patients and analyze them correctly. Methods: The blood culture results of patients at Gachon University Gil Medical Center from 2016 to 2020 were analyzed retrospectively to determine the bacterial distributions and antibiotic resistance patterns. Duplicates were eliminated by including only the first isolate of each species per patient. Results: Escherichia coli (27.1%) was the most commonly isolated bacterium from blood cultures, followed by Klebsiella pneumoniae (10.1%) and Staphylococcus aureus (8.6%). The methicillin resistance rate of S. aureus was 49.2%, and the vancomycin resistance rate of Enterococcus faecium was 39.5%; with no significant changes over the study period. The cefotaxime, ciprofloxacin, and ertapenem resistance rates of E. coli were 35.0%, 46.8%, and 0.7%, respectively. Seventeen carbapenem-resistant E. coli strains were isolated, of which 11 produced carbapenemase. The cefotaxime, ciprofloxacin, and ertapenem resistance rates of K. pneumoniae were 29.5%, 31.7%, and 5.4%, respectively. Forty-eight carbapenem-resistant K. pneumoniae strains were isolated, of which 37 produced carbapenemase. The imipenem resistance rates of Acinetobacter baumannii and Pseudomonas aeruginosa were 72.3% and 23.4%, respectively. Conclusion: In the blood culture results from 2016 to 2020, the isolation frequency of E. coli, K. pneumoniae, and E. faecium showed an increasing trend, whereas that of S. aureus was stable. Over the 5 year study period, the ciprofloxacin resistance rate of E. coli and P. aeruginosa and ampicillin/sulbactam resistance rate of A. baumannii significantly increased.

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

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

Carbapenem resistant Enterobacterales in the United Arab Emirates: a retrospective analysis from 2010 to 2021.

Carbapenem-resistant Enterobacterales (CRE) are spreading in the United Arab Emirates (UAE) where their dissemination is facilitated by international travel, trade, and tourism. The objective of this study is to describe the longitudinal changes of CRE as reported by the national AMR surveillance system of the UAE. In this study, we retrospectively describe CRE isolated from 317 surveillance sites, including 87 hospitals and 230 centers/clinics from 2010 to 2021. The associated clinical, demographic, and microbiological characteristics are presented by relying on the UAE national AMR surveillance program. Data was analyzed using WHONET microbiology laboratory database software (http://www.whonet.org). A total of 14,593 carbapenem resistant Enterobacterales were analyzed, of which 48.1% were carbapenem resistant Klebsiella pneumoniae (CRKp), 25.1% carbapenem resistant Escherichia coli (CREc), and 26.8% represented 72 other carbapenem resistant species. Carbapenem resistant strains were mostly associated with adults and isolated from urine samples (36.9% of CRKp and 66.6% of CREc) followed by respiratory samples (26.95% for CRKp) and soft tissue samples (19.5% for CRKp). Over the studied period carbapenem resistance rates remained high, especially in K. pneumoniae, and in 2021 were equivalent to 67.6% for imipenem, 76.2% for meropenem, and 91.6% for ertapenem. Nevertheless, there was a statistically significant decreasing trend for imipenem and meropenem resistance in Klebsiella species (p < 0.01) while the decrease in ertapenem resistance was non-significant. Concerning E. coli, there was a statistically significant decreasing trend for meropenem and imipenem resistance over the 12 years, while ertapenem resistance increased significantly with 83.8% of E. coli exhibiting ertapenem resistance in 2021. Resistance rates to ceftazidime and cefotaxime remained higher than 90% (in 2021) for CRKp and cefotaxime rates increased to 90.5% in 2021 for CREc. Starting 2014, resistance to colistin and tigecycline was observed in carbapenem resistant Enterobacterales. CRE were associated with a higher mortality (RR: 6.3), admission to ICU (RR 3.9), and increased length of stay (LOS; 10 excess inpatient days per CRE case). This study supports the need to monitor CRE in the UAE and draws attention to the significant increase of ertapenem resistance in E. coli. Future surveillance analysis should include a genetic description of carbapenem resistance to provide new strategies.

Platinum Nanoparticles Prevent the Resistance of Pseudomonas aeruginosa to Ciprofloxacin and Imipenem: Mechanism Insights.

Metal nanoparticles (MNPs) have recently gained extensive attention due to their broad-spectrum prospect, particularly in biomedical application. Here, we reveal that long-term exposure to platinum nanoparticles (Pt NPs) increases the susceptibility of Pseudomonas aeruginosa PAO1 to imipenem and ciprofloxacin. We exposed PAO1 to Pt NPs (a series of doses, varying from 0.125 to 35 μg/mL) for 60 days and characterized the evolved strains (ES) and compared with wild type (WT) to understand the mechanism of heightened sensitivity. We found that overexpression of oprD and downregulation of mexEF-oprN facilitate the intracellular accumulation of antibiotic, thus increasing susceptibility. Furthermore, loss-of-function mutations were discovered in regulators lasR and mexT. Cloning intact lasR from wild-type (WT) into ES slightly improves imipenem resistance. Strikingly, cloning mexT from WT into ES reverts the imipenem and ciprofloxacin resistance to the original level. Briefly, the increase of membrane permeability controlled by mexT made PAO1 greatly susceptible to imipenem and ciprofloxacin, and the decrease of quorum sensing mediated by lasR made PAO1 slightly susceptible to imipenem. Overall, these results reveal an antibiotic susceptibility mechanism from prolonged exposure to MNPs, which provides a promising approach to prevent antibiotic resistance.

Bacterial profile of surgical site infection and antimicrobial resistance patterns in Ethiopia: a multicentre prospective cross-sectional study

BackgroundGlobally, surgical site infections (SSI) are the most commonly reported healthcare-associated infections.MethodsA multicentre study was conducted among patients who underwent surgical procedures at four hospitals located in Northern (Debre Tabor), Southern (Hawassa), Southwest (Jimma), and Central (Tikur Anbessa) parts of Ethiopia. A total of 752 patients clinically studied for surgical site infection were enrolled. The number of patients from Debre Tabor, Hawassa, Jimma, and Tikur Anbessa, hospitals was 172, 184, 193, and 203, respectively. At each study site, SSI discharge culture was performed from all patients, and positive cultures were characterized by colony characteristics, Gram stain, and conventional biochemical tests. Each bacterial species was confirmed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF). An antimicrobial susceptibility test (AST) was done on Mueller–Hinton agar using the disk diffusion method. Logistic regression analysis was used to assess associations of dependent and independent variables. A p-value < 0.05 was considered statistically significant. Data were analysed using STATA 16 software.ResultsAmong 752 wound discharge cultures performed, 65.5% yielded growth. Among these, 57.9% and 42.1% were Gram-negative and Gram-positive isolates, respectively. In this study, a total of 494 bacteria were isolated; Staphylococcus aureus (31%), Escherichia coli (20.7%), and Klebsiella pneumoniae (9.8%) were the most common. Rare isolates (0.8% each) included Raoultella ornithinolytica, Stenotrophomonas maltophilia, Alcalignes faecalis, Pantoea ecurina, Bacillus flexus, and Paenibacillus tylopili. Enterobacteriaceae showed high levels of resistance to most of the tested antibiotics but lower levels of ertapenem (32.9%), amikacin (24.3%), imipenem (20.3%), and meropenem (17.6%) resistance. Multidrug-resistant (MDR) frequency of Enterobacteriaceae at Debre Tabor, Hawassa, Jimma, and Tikur Anbessa hospitals was 84.5%, 96.5%, 97.3%, and 94%, respectively. Ages ≥ 61 years (AOR = 2.83, 95% CI: 1.02–7.99; P 0.046), prolonged duration of hospital stay (AOR = 4.15, 95% CI: 2.87–6.01; P 0.000), history of previous antibiotics use (AOR = 2.83, 95% CI: 1.06–2.80; P 0.028), history of smoking (AOR = 2.35, 95% CI: 1.44–3.83; P 0.001), emergency surgery (AOR = 2.65, 95% CI: 1.92–3.66; P 0.000), and duration of operation (AOR = 0.27, 95% CI: 0.181–0.392; P 0.000) were significant risk factors.ConclusionThe most prevalent isolates from Gram-positive and Gram-negative bacteria across all hospitals were S. aureus and E. coli, respectively. Many newly emerging Gram-negative and Gram-positive bacteria were identified. Variation between hospitals was found for both SSI etiology type and MDR frequencies. Hence, to prevent the emergence and spread of MDR bacteria, standard bacteriological tests and their AST are indispensable for effective antimicrobial stewardship.

Effect of piperine on the inhibitory potential of MexAB-OprM efflux pump and imipenem resistance in carbapenem-resistant Pseudomonas aeruginosa

The escalating prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant threat to global public health through the spread of its ‘high-risk’ clones. Immediate and decisive research into antimicrobial agents against CRPA is crucial for the development of effective measures and interventions. Overexpression of the MexAB-OprM efflux pump is one of the major mechanisms of CRPA. Since the active efflux of antibacterial agents plays a significant role in mediating drug resistance in CRPA, the inhibition of efflux pumps has become a promising strategy to restore antibacterial potency. Piperine (PIP) has been proven to be a promising efflux pump inhibitor in some bacteria. However, there are no studies on whether PIP can act as a potential efflux pump inhibitor in CRPA. The present study aimed to identify the antibacterial activity of PIP against CRPA and to evaluate the effect on the MexAB-OprM efflux pump. Molecular docking was used to analyze the possible interaction of PIP with the proteins of the MexAB-OprM efflux pump in CRPA. The effect of PIP on the expression of the MexAB-OprM efflux pump was investigated by real-time quantitative PCR (qPCR) and ethidium bromide accumulation efflux assay. The effect of PIP on CRPA imipenem (IPM) resistance was investigated by the checkerboard dilution method. The results demonstrated that PIP exhibited the lowest binding affinity of −9.1 kcal towards efflux pump proteins. A synergistic effect between PIP and IPM on CRPA was observed. More importantly, PIP effectively hindered the efflux of ethidium bromide and IPM by up-regulating MexR gene expression while down-regulating MexA, MexB, and OprM gene expressions. In conclusion, PIP could enhance the antibacterial activity of IPM by inhibiting the MexAB-OprM efflux pump. Our work proved that PIP had the potential to be an efflux pump inhibitor of CRPA.

Direct prediction of carbapenem resistance in Pseudomonas aeruginosa by whole genome sequencing and metagenomic sequencing.

Carbapenem resistance is a major concern in the management of antibiotic-resistant Pseudomonas aeruginosa infections. The direct prediction of carbapenem-resistant phenotype from genotype in P. aeruginosa isolates and clinical samples would promote timely antibiotic therapy. The complex carbapenem resistance mechanism and the high prevalence of variant-driven carbapenem resistance in P. aeruginosa make it challenging to predict the carbapenem-resistant phenotype through the genotype. In this study, using whole genome sequencing (WGS) data of 1,622 P. aeruginosa isolates followed by machine learning, we screened 16 and 31 key gene features associated with imipenem (IPM) and meropenem (MEM) resistance in P. aeruginosa, including oprD(HIGH), and constructed the resistance prediction models. The areas under the curves of the IPM and MEM resistance prediction models were 0.906 and 0.925, respectively. For the direct prediction of carbapenem resistance in P. aeruginosa from clinical samples by the key gene features selected and prediction models constructed, 72 P. aeruginosa-positive sputum samples were collected and sequenced by metagenomic sequencing (MGS) based on next-generation sequencing (NGS) or Oxford Nanopore Technology (ONT). The prediction applicability of MGS based on NGS outperformed that of MGS based on ONT. In 72 P. aeruginosa-positive sputum samples, 65.0% (26/40) of IPM-insensitive and 63.2% (24/38) of MEM-insensitive P. aeruginosa were directly predicted by NGS-based MGS with positive predictive values of 0.897 and 0.889, respectively. By the direct detection of the key gene features associated with carbapenem resistance of P. aeruginosa, the carbapenem resistance of P. aeruginosa could be directly predicted from cultured isolates by WGS or from clinical samples by NGS-based MGS, which could assist the timely treatment and surveillance of carbapenem-resistant P. aeruginosa.

Bla KPC-2 overexpression and bla GES-5 carriage as major imipenem/relebactam resistance mechanisms in Pseudomonas aeruginosa high-risk clones ST463 and ST235, respectively, in China.

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5-carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage.

Global genomic epidemiology of chromosomally mediated non-enzymatic carbapenem resistance in Acinetobacter baumannii: on the way to predict and modify resistance.

Although carbapenemases are frequently reported in resistant A. baumannii clinical isolates, other chromosomally mediated elements of resistance that are considered essential are frequently underestimated. Having a wide substrate range, multidrug efflux pumps frequently underlie antibiotic treatment failure. Recognizing and exploiting variations in multidrug efflux pumps and penicillin-binding proteins (PBPs) is an essential approach in new antibiotic drug discovery and engineering to meet the growing challenge of multidrug-resistant Gram-negative bacteria. A total of 980 whole genome sequences of A. baumannii were analyzed. Nucleotide sequences for the genes studied were queried against a custom database of FASTA sequences using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) system. The correlation between different variants and carbapenem Minimum Inhibitory Concentrations (MICs) was studied. PROVEAN and I-Mutant predictor suites were used to predict the effect of the studied amino acid substitutions on protein function and protein stability. Both PsiPred and FUpred were used for domain and secondary structure prediction. Phylogenetic reconstruction was performed using SANS serif and then visualized using iTOL and Phandango. Exhibiting the highest detection rate, AdeB codes for an important efflux-pump structural protein. T48V, T584I, and P660Q were important variants identified in the AdeB-predicted multidrug efflux transporter pore domains. These can act as probable targets for designing new efflux-pump inhibitors. Each of AdeC Q239L and AdeS D167N can also act as probable targets for restoring carbapenem susceptibility. Membrane proteins appear to have lower predictive potential than efflux pump-related changes. OprB and OprD changes show a greater effect than OmpA, OmpW, Omp33, and CarO changes on carbapenem susceptibility. Functional and statistical evidence make the variants T636A and S382N at PBP1a good markers for imipenem susceptibility and potential important drug targets that can modify imipenem resistance. In addition, PBP3_370, PBP1a_T636A, and PBP1a_S382N may act as potential drug targets that can be exploited to counteract imipenem resistance. The study presents a comprehensive epidemiologic and statistical analysis of potential membrane proteins and efflux-pump variants related to carbapenem susceptibility in A. baumannii, shedding light on their clinical utility as diagnostic markers and treatment modification targets for more focused studies of candidate elements.

Marine resistome of a temperate zone: Distribution, diversity, and driving factors across the trophic gradient

Marine and ocean environments are the most widespread habitats in the world but are still the least studied from the aspect of antibiotic resistance. The indigenous and tetracycline (TET)- and sulfamethoxazole (SXT)-resistant planktonic bacterial communities were simultaneously investigated for the first time along a trophic gradient of a temperate zone, regarding their taxonomic and functional structures as well as biotic and abiotic factors affecting their dynamics as vehicles of antibiotic resistance genes (ARGs), thus impacting the ARGs distribution at seasonal and spatial scales.A total of 80 microbiomes, recovered seasonally from bottom layer and surface waters along a 68-km transect from wastewater-impacted estuary to coastal and pristine open sea in the central Adriatic (Mediterranean Sea), were analysed using 16S rRNA amplicon sequencing, PICRUSt2 bioinformatics and extensive biostatistics. Eighty-one bacterial phyla were identified, with majority (n = 49) in summer when communities were found to be more species enriched across the gradient. Microbial diversity was more site-specific and pronounced in surface microbiomes in winter. Nevertheless, both richness and community diversity decreased with distance from the coast. Although the microbiomes from human-influenced sites significantly differed from those in oligotrophic offshore area, Proteobacteria were still the most abundant phylum during both seasons at the surface and seabed along the gradient, and the major contributors to the marine resistome regarding native and TET- and SXT-resistant microbial communities. Resistome structure was more diverse in winter, whereas peptide, vancomycin and multidrug resistance modules predominated regardless of season, trophic status, or antibiotic. However, multidrug, beta-lactam resistance modules as well as macrolide, phenicol, aminoglycoside, and particularly imipenem resistance genes were much more frequent in winter, suggesting that the diversity of indigenous resistomes is highly dependent on seasonal variations of the water column, driven by thermohaline stratification and nutrients. Moreover, several pathogenic genera stood out as important carriers of multiple resistance traits in TET- and SXT-related resistomes in both seasons, particularly Acinetobacter, Vibrio, Bacillus and Pseudomonas, beside which Proteus, Serratia and Bacteroides prevailed in native resistomes.This study evidenced seasonal and spatial variations of the marine microbiome and resistome and their driving forces along the trophic gradient, providing a comprehensive insight into the diversity and distribution of antibiotic resistance in the marine ecosystem of a temperate zone.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P.aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

Risk Factors and Outcome of Gram-Negative Bloodstream Infection in Living-Donor Renal Transplant Recipients: A Case-Control Study From Pakistan.

Gram-negative rods are the most common cause of bloodstream infection in renal transplant recipients. Acute rejection, urologic abnormalities, and ureteral stents are risk factors. Graft dysfunction is independently associated with gram-negative rod bloodstream infection. Our aim is to investigate the incidence, risk factors, and outcome among living donor renal transplant recipients from Pakistan. In this case-control study, we reviewed the medical records until June 2021 of renal transplant recipients seen from 2015 to 2019 for gram negative bacteremia. For every case, controls were matched by age, date of transplant, and sex. Demographics, risk factors, graft function, and mortality were compared. Clinical features, immunosuppression, source of blood stream infection, and microbiology were noted in cases. Of 1677 renal transplant recipients, 44 developed gram negative bacteremia. The incidence was 5.9 per 1000 person-years. Median time since transplant was 5 months. The most common source was urinary tract infection. On univariate analysis, antithymocyte globulin, urinary tract infection, and recurrent urinary tract infections were associated with gram negative bacteremia. On multivariate analysis, urinary tract infection (adjusted odds ratio = 3.46; 95% CI, 1.27-9.37) and recurrent urinary tract infections (adjusted odds ratio = 4.03; 95% CI, 1.15-14.15) were significant risk factors. We found no difference in 30-day mortality and estimated glomerular filtration rate on last follow-up between cases and controls. Kaplan-Meier survival curves showed significant differences in graft survival in patients with gram negative bacteremia. Escherichia coli was the most common organism, with 75% ceftriaxone and 13% imipenem resistance. The most significant risk factor for gram negative rod bloodstream infection was recurrent urinary tract infections. Timely treatment and prevention of recurrent urinary tract infections areimperative for prevention of gram negative bacteremia.

Emergence of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacterales from water sources: An impending public health challenge in Adamawa-north senatorial zone, Nigeria

Background: The emergence of New Delhi metallo-beta-lactamase-1 (NDM-1) among Enterobacterales in water sources has raised a major public health concern and constitute critical threat to human health as these organisms exhibits high level of resistance to available potent antibiotics. The aim of this study is to detect the presence of NDM-1 gene among carbapenem resistant Enterobacterales (CRE) isolates from water sources.&#x0D; Methodology: A total of 256 water samples were collected from randomly selected hand-dug wells (128 samples) and river/stream (128 samples) for each of dry and rainy seasons in four out of the five local government areas (LGAs) of Adamawa-north senatorial zone, Nigeria. The water samples were filtered using membrane filtration technique and the filters introduced into appropriate bacteriologic media for bacterial growth. The bacterial isolates recovered were identified by both phenotypic and molecular protocols. Phenotypic carbapenem (imipenem) resistance was determined by disc diffusion test, blaNDM-1 gene was detected by specific polymerase chain reaction (PCR) test, and plasmid DNA was extracted and electrophoresed by standard procedure.&#x0D; Results: Of the 256 water samples analyzed for bacteria growth, 300 bacterial isolates of the order Enterobacterales were recovered. Of these, only 45 (12.6%) isolates were phenotypically resistant to carbapenem (imipenem) antibiotic and blaNDM-1 gene was detected in 30 (66.7%) of these. While blaNDM-1 gene was detected in all the isolates of Klebsiella oxytoca, Klebsiella variicola, Enterobacter aerogenes, Enterobacter hormaechei, Enterobacter asburiae, Citrobacter freundii, and Morganella morganii that were resistant to imipenem, other isolates harbored blaNDM-1 gene in varying proportion. Most of the isolates positive for blaNDM-1 also harbored R-plasmids.&#x0D; Conclusion: Emergence of carbapenem resistance mediated by NDM-1 gene in Enterobacterales isolated from water sources constitutes an emerging public health challenge with potential transmission to humans, thereby complicating the treatment of infections caused by these resistant pathogens in man. As such, the urgent need for antimicrobial surveillance and stewardship is of utmost importance.

Antimicrobial susceptibility of Bacteroides fragilis group organisms in Hong Kong, 2020–2021

ObjectivesThis retrospective study analyzed the susceptibility levels of Bacteroides fragilis group (BFG) in a hospital-based laboratory where disk diffusion test (DDT) was routinely performed. Isolates non-susceptible to imipenem and metronidazole by DDT were further investigated using a gradient method. MethodsThe DDT and MIC susceptibility data of clindamycin, metronidazole, moxifloxacin and imipenem obtained on Brucella blood agar for 1264 non-duplicated isolates during 2020–2021 were analyzed. Species identification was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and 16S rRNA sequencing. Interpretative agreement of DDT results using the 2015 EUCAST tentative and 2021 CA-SFM breakpoints was compared against MIC as the reference. ResultsThe dataset included 604 B. fragilis (483 division I, 121 division II isolates), 415 non-fragilis Bacteroides, 177 Phocaeicola and 68 Parabacteroides. Susceptibility rates for clindamycin (22.1–62.1%) and moxifloxacin (59.9–80.9%) were low and many had no inhibition zones. At the EUCAST and CA-SFM breakpoints, 83.0 and 89.4% were imipenem-susceptible, and 89.6% and 97.4 were metronidazole-susceptible. MIC testing confirmed 11.4% and 2.8% isolates as imipenem-non-susceptible and metronidazole-resistant, respectively. Significant numbers of false-susceptibility and/or false-resistance results were observed at the CA-SFM breakpoint but not the EUCAST breakpoint. Higher rates of imipenem and/or metronidazole resistance were detected in B. fragilis division II, B. caccae, B. ovatus, B. salyersiae, B. stercoris and Parabacteroides. Co-resistance to imipenem and metronidazole was detected in 3 B. fragilis division II isolates. ConclusionsThe data demonstrated emerging BFG resistance to several important anti-anaerobic antibiotics and highlights the importance of anaerobic susceptibility testing in clinical laboratories to guide therapy.

Phenotypic characterization and epidemiology of extended-spectrum β-lactamase-producing Enterobacteriaceae strains from urinary tract infections in Garoua, Cameroon.

The emergence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) is causing increased morbidity and mortality around the world as a result of therapeutic failures. ESBL-E are priority pathogens due to their multidrug resistance (MDR). In Northern Cameroon, ESBL-producing bacteria, particularly in urinary tract infections (UTIs), are being increasingly isolated. This study aimed to retrospectively determine the prevalence of multi-drug resistant ESBL strains isolated from UTIs in Northern Cameroon and to evaluate the effectiveness of the ATB UR Gallery of BioMérieux in diagnosing ESBL-E in clinical settings. Standard microbiology protocols and statistical tools were utilized to identify ESBL-producing bacteria and characterize their phenotypic susceptibility and resistance profiles in the study population. Out of the 144 enterobacteria isolates successfully cultured, 59 (41%) were identified as MDR strains. The ATB UR EU gallery identified 33 (23%) multi-drug resistant ESBL-producing strains, while the double synergy test identified 35 strains without disc reconciliation and 38 strains after reconciliation. The most prevalent ESBL-E isolate was Escherichia coli, accounting for 77.1% of the isolates, followed by Klebsiella pneumoniae (20%) and Enterobacter aerogenes (2.9%). Additionally, the study revealed the emergence of Imipenem resistance (5.7%), a critical last-resort antibiotic. However, all ESBL strains were sensitive to Fosfomycin (FSF/FOS), demonstrating its potential as an effective therapeutic option. Moreover, 37% of the ESBL producers exhibited co-resistance to over 20 different antibiotics. This study provides valuable insights into the prevalence and susceptibility patterns of ESBL-E associated with UTIs in Northern Cameroon. These insights emphasizes the importance of implementing appropriate treatment guidelines and antimicrobial stewardship measures to mitigate the spread and impact of MDR ESBL-producing strains on public health.