Carbapenem Resistance Research Articles

Non-thermal obliteration of critically ranked carbapenem-resistant Acinetobacter baumannii and its resistance gene in a batch atmospheric plasma reactor

Wastewater treatment plants (WWTPs) have been implicated as direct key reservoir of both antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) associated with human infection, as high concentrations of ARBs and ARGs have been detected in recycled hospital wastewater. Among the ARBs, the carbapenem-resistant Acinetobacter baumannii has been ranked as priority 1 (critical) pathogen by the World Health Organization (WHO), due to its overwhelming burden on public health. Therefore, this study is aimed at investigating non-thermal plasma (NTP) technology as an alternative disinfection step to inactivate this bacterium and its ARGs. Culture-based method and PCR were employed in confirming the carbapenem resistance gene blaNDM-1 in A. baumannii (BAA 1605). Suspension of carbapenem-resistant A. baumannii (24 h culture) was prepared from the confirmed isolate and subjected to plasma treatment at varying time intervals (3 min, 6 min, 9 min, 12 min, and 15 min) in triplicates. The plasma-treated samples were evaluated for re-growth and the presence of the resistance gene. The treatment resulted in a 1.13 log reduction after 3 min and the highest log reduction of ≥ 8 after 15 min, and the results also showed that NTP was able to inactivate the blaNDM-1 gene. The log reduction and gel image results suggest that plasma disinfection has a great potential to be an efficient tertiary treatment step for WWTPs.

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/ H 30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P -value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing bla CTX-M-15 copy numbers via modular, insertion sequence 26 (IS 26 ) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/ H 30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

Predictive modeling of mortality in carbapenem-resistant Acinetobacter baumannii bloodstream infections using machine learning.

Acinetobacter baumannii, a notable drug-resistant bacterium, often induces severe infections in healthcare settings, prompting a deeper exploration of treatment alternatives due to escalating carbapenem resistance. This study meticulously examined clinical, microbiological, and molecular aspects related to in-hospital mortality in patients with carbapenem-resistant A. baumannii (CRAB) bloodstream infections (BSIs). From 292 isolates, 153 cases were scrutinized, reidentified through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and evaluated for antimicrobial susceptibility and carbapenemase genes via multiplex polymerase chain reaction (PCR). Utilizing supervised machine learning, the study constructed models to predict 14- and 30-day mortality rates, revealing the Naïve Bayes model's superior specificity (0.75) and area under the curve (0.822) for 14-day mortality, and the Random Forest model's impressive recall (0.85) for 30-day mortality. These models delineated eight and nine significant features for 14- and 30-day mortality predictions, respectively, with "septic shock" as a pivotal variable. Additional variables such as neutropenia with neutropenic days prior to sepsis, mechanical ventilator support, chronic kidney disease, and heart failure were also identified as ranking features. However, empirical antibiotic therapy appropriateness and specific microbiological data had minimal predictive efficacy. This research offers foundational data for assessing mortality risks associated with CRAB BSI and underscores the importance of stringent infection control practices in the wake of the scarcity of new effective antibiotics against resistant strains. The advanced models and insights generated in this study serve as significant resources for managing the repercussions of A. baumannii infections, contributing substantially to the clinical understanding and management of such infections in healthcare environments.

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

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

Genomic characterisation of a multidrug-resistant Klebsiella pneumoniae co-harbouring blaNDM-1, blaKPC-2, and tet(A) isolated from the bloodstream infections of patients

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A) and the subsequent analysis of its genomic features. Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis. A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS26, ISAba125, and IS26, respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46. The present study identified the occurrence of a rare plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A), and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention.

Ceftazidime-avibactam resistance determination in carbapenem-resistant Klebsiella pneumoniae infections before its use in practice.

To ensure the appropriate usage of ceftazidime-avibactam (CAZ-AVI), recently introduced in our hospital, we aimed to determine susceptibility rates, enzyme analysis, and clonal relationship among strains, together with clinical data. Between June 1 and September 30, 2021, demographic and microbiological data of the patients were recorded. In the obtained samples, meropenem and colistin minimal inhibitory concentration (MIC) levels, carbapenem resistance genes, and the clonal relationship were studied by molecular methods. CAZ-AVI was not used in any of the patients. 140 carbapenem-resistant Klebsiella pneumoniae were isolated from 57 patients. Resistance to CAZ-AVI was found in 76 (54.3%) strains. Out of 57 patients, 31 (54.4%) isolates could be reached. Meropenem MIC level was ≥ 32 µg/mL in 26 (83.9%), and colistin MIC level was ≥ 4 µg/mL in 17 (54.8%) isolates. Enzyme analysis revealed NDM in 20 (64.5%), OXA-48 in 17 (54.8%), and KPC in seven (22.6%). NDM + OXA-48 was determined in 10 (32.2%) strains. NDM was determined in all CAZ-AVI resistant strains, OXA-48 in 16.1% (2/5) strains. Seven genotypes were detected. The largest cluster was genotype 3 clusters (11 isolates). Of 31 patients, 22 (71.0%) died. CAZ-AVI was susceptible in one of the patients who survived and four who died. Before using a new antibiotic, each center should determine the basal data and phenotypic/genotypic resistance ratios specific to that antibiotic. While a high NDM rate and low CAZ-AVI sensitivity limit the use of the drug in our center, it is clear that CAZ-AVI use in sensitive strains will decrease mortality.

Combined Use of Phenotypic Screening and of a Novel Commercial Assay (REALQUALITY Carba-Screen) for the Rapid Molecular Detection of Carbapenemases: A Single-Center Experience.

Carbapenem resistance is a serious public health threat, causing numerous deaths annually primarily due to healthcare-associated infections. To face this menace, surveillance programs in high-risk patients are becoming a widespread practice. Here we report the performance of the combined use of a recently approved commercial multiplex real-time PCR assay (REALQUALITY Carba-Screen kit) with conventional phenotypic screening. In this three-month study, 479 rectal swabs from 309 patients across high-risk units were evaluated by combining the two approaches. Although the molecular assay showed a higher positivity rate than phenotypic screening (7.1% vs. 5%), it should be noted that the molecular method alone would have missed eight carbapenem-resistant isolates, while using only phenotypic screening would not have detected sixteen isolates. This demonstrates the complementary strengths of each method. Our study confirms the need for a combined approach to maximize the possible clinical impact of this kind of screening, ensuring a more comprehensive detection of resistant strains.

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.

Therapeutic Phage Candidates for Targeting Prevalent Sequence Types of Carbapenem-Resistant Escherichia coli.

Carbapenem-resistant Escherichia coli (CREC) is a global threat to public health; therefore, alternative treatment options are urgently needed. Bacteriophages have emerged as promising candidates for combating CREC infections. This study aimed to investigate the genetic basis of phage sensitivity in CREC by evaluating carbapenem resistance among multidrug-resistant (MDR) E. coli isolated in Daegu, South Korea and analyzing their sequence types (STs) with phage susceptibility spectra. Among the 60 MDR E. coli isolates, 80.4% were identified as CREC, with 77.0% demonstrating resistance to imipenem and 66.6% to meropenem. Moreover, 70 lytic E. coli bacteriophages were isolated from hospital sewage water and evaluated against those 60 E. coli isolates. The phages exhibited lytic activity of 33%-60%, with average titers ranging from 5.6 × 1012 to 2.4 × 1013 PFU/mL (Plaque-Forming Unit). Furthermore, multilocus sequence typing (MLST) analysis of the bacterial isolates revealed 14 distinct STs, mostly belonging to ST131, ST410, and ST648. Notably, the phage susceptibility spectra of ST73, ST13003, ST648, ST2311, ST167, ST405, ST607, ST7962, and ST131 were significantly different. Thus, the isolated phages can effectively lyse CREC isolates, particularly those with clinically dominant STs. Conversely, ST410 exhibited a 14.2%-87.14% susceptibility spectrum, whereas ST1139, ST1487, ST10, and ST206 did not lyse, suggesting the presence of more resistant STs. Future studies are warranted to identify the reasons behind this resistance and address it. Ultimately, this study will aid in developing focused treatments to address these pressing global health issues.

Plasmid-Mediated Spread of Carbapenem Resistance in Enterobacterales: A Three-Year Genome-Based Survey.

The worldwide emergence and dissemination of carbapenem-resistant Gram-negative bacteria (CRGNB) is a challenging problem of antimicrobial resistance today. Outbreaks with CRGNB have severe consequences for both the affected healthcare settings as well as the patients with infection. Thus, bloodstream infections caused by metallo-ß-lactamase-producing Enterobacterales can often have clinical implications, resulting in high mortality rates due to delays in administering effective treatment and the limited availability of treatment options. The overall threat of CRGNB is substantial because carbapenems are used to treat infections caused by ESBL-producing Enterobacterales which also exist with high frequency within the same geographical regions. A genome-based surveillance of 589 CRGNB from 61 hospitals across the federal state Hesse in Germany was implemented using next-generation sequencing (NGS) technology to obtain a high-resolution landscape of carbapenem-resistant isolates over a three-year period (2017-2019). The study examined all reportable CRGNB isolates submitted by participating hospitals. This included isolates carrying known carbapenemases (435) together with carbapenem-resistant non-carbapenemase producers (154). Predominant carbapenemase producers included Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Acinetobacter baumannii. Over 80% of 375 carbapenem-resistant determinants including KPC-, NDM-, VIM- and OXA-48-like ones detected in 520 Enterobacterales were plasmid-encoded, and half of these were dominated by a few incompatibility (Inc) types, viz., IncN, IncL/M, IncFII and IncF(K). Our results revealed that plasmids play an extraordinary role in the dissemination of carbapenem resistance in the heterogeneous CRGNB population. The plasmids were also associated with several multispecies dissemination events and local outbreaks throughout the study period, indicating the substantial role of horizontal gene transfer in carbapenemase spread. Furthermore, due to vertical and horizontal plasmid transfer, this can have an impact on implant-associated infections and is therefore important for antibiotic-loaded bone cement and drug-containing devices in orthopedic surgery. Future genomic surveillance projects should increase their focus on plasmid characterization.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

High prevalence of carbapenem-resistant Enterobacterales (CRE) in human samples from Nigeria: A systematic review and meta-analysis

ObjectivesThe rise in Carbapenem-resistant Enterobacterales (CRE) is perturbing. To curb the menace of CRE, a comprehensive understanding of its prevalence and epidemiology is crucial. As varying reports abound, the true prevalence of CRE in Nigeria remains unknown. Here, we conducted a systematic review and meta-analysis following standard guidelines to assess the situation of CRE in Nigeria. MethodsWe searched electronic databases including Pubmed, ScienceDirect, Scopus, Web of Science, and Google Scholar for articles providing information on CRE in Nigeria. The data gathered were analyzed using OpenMeta Analyst and Comprehensive Meta-Analysis software. The random-effect model was employed to calculate pooled resistance to carbapenem antibiotics. ResultsFrom 321 retrieved records, 57 were finally included. The studies were predominantly from the South-West region (n = 19). Escherichia coli and Klebsiella pneumoniae were the most frequently tested Enterobacterales among the included studies. The pooled prevalence estimate for imipenem resistance among CRE was 11.2 % (95 % CI: 7.9–15.7). Meropenem resistance had an estimate of 13.5 % (95 % CI: 9.1–19.6), whereas ertapenem and doripenem were estimated at 17.0 % (95 % CI: 9.9–27.7) and 37.9 % (95 % CI: 15.0–67.8), respectively. High heterogeneity (I2>85 %, p < 0.001) was observed for the estimates. The highest resistance rate to imipenem (28.4 %), meropenem (37.2 %) and ertapenem (46.5 %) were observed for the South-South region. Based on specific CRE genera, Morganella sp. was the most resistant (37.0 %) while Escherichia sp. was the least (9.4 %). Our analyses also revealed a progressive increase in resistance to carbapenem antibiotics over the years. ConclusionThis study highlights carbapenem resistance as a concern in Africa's most populous nation, underscoring the need for proactive measures to address and mitigate the threat of CRE.

Carbapenem-resistant Acinetobacter baumannii infections among diabetic and non-diabetic patients and possible effective combination treatments

BackgroundCarbapenems are one of the most noteworthy choices for treating multidrug-resistant Acinetobacter baumannii (A. baumannii). Currently, carbapenem-resistant A. baumannii (CRAB) represents a healthcare problem worldwide, particularly among diabetic patients who are more susceptible to microbial infections. The aim of this study was to investigate the differences in antibiotic susceptibility profiles, the abundance of carbapenem resistance genes across A. baumannii-infected diabetic and non-diabetic patients, and the antimicrobial activity of different antibiotic combinations on highly resistant isolates.MethodsData of 99 A. baumannii-infected patients were collected during the period from 2018 to 2022 and categorized according to patients’ diabetes status into either diabetic or non-diabetic group. A total of 45 A. baumannii isolates were collected during 2021 and 2022 from the main hospital laboratory to be reidentified and genetically confirmed. Antibiotic susceptibility, including carbapenems, was determined using disc agar diffusion and broth microdilution methods. The isolates were screened for OXA-23, GES, VIM, and NDM carbapenem-resistant genes. Five antibiotic combinations were assessed using the double-disk synergy and checkerboard methods.ResultsThe findings of the current study revealed that multidrug resistance increased gradually, from 56% in 2018 to 95.6% in 2022. Moreover, CRAB increased among diabetics and non-diabetics. Resistance rates of imipenem, meropenem, and doripenem reached 68.8%, 61.8%, and 47.4% in diabetics and 97.9%, 83.3%, and 50% in non-diabetics, respectively. The VIM gene was the most prevalent gene with prevalence rates of 100% and 96.15% in diabetics and non-diabetics, respectively. Moreover, all A. baumannii isolates carried at least two of the selected carbapenem-resistant genes. Across the different used combinations, only the tigecycline-meropenem combination showed synergistic activity in 50% of diabetic and 66.7% of non-diabetic isolates.ConclusionsAn increased carbapenem resistance was observed among A. baumannii-infected individuals, both diabetic and non-diabetic. The MEM/TCG combination was the only one that showed synergistic or additive effects against highly resistant isolates making it a viable alternative treatment option.

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

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

Genomic Characterization of a Clinical NDM-1-Producing Klebsiella michiganensis from Brazil.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

Risk factors for bloodstream infections due to carbapenem-resistant Enterobacterales: a nested case-control-control study.

Carbapenem-resistant Enterobacterales (CRE) bloodstream infections (BSIs) are a major threat to patients. To date, data on risk factors have been limited, with low internal and external validity. In this multicentre study, risk factors for CRE BSI were determined by comparison with two control groups: patients with carbapenem-susceptible Enterobacterales (CSE) BSI, and patients without Enterobacterales infection (uninfected patients). A multicentre, case-control-control study was nested in a European prospective cohort study on CRE (EURECA). CRE BSI:CSE BSI matching was 1:1, CRE BSI:Uninfected patients matching was 1:3, based on hospital, ward and length of stay. Conditional logistic regression was applied. From March 2016 to November 2018, 73 CRE BSIs, 73 CSE BSIs and 219 uninfected patients were included from 18 European hospitals. For CRE versus CSE BSI, previous CRE colonization/infection [incidence rate ratio (IRR) 7.32; 95% CI 1.65-32.38) increased the risk. For CRE versus uninfected controls, independent risk factors included: older age (IRR 1.03; 95% CI 1.01-1.06), patient referral (long-term care facility: IRR 7.19; 95% CI 1.51-34.24; acute care hospital: IRR 5.26; 95% CI 1.61-17.11), previous colonization/infection with other MDR organisms (MDROs) (IRR 9.71; 95% CI 2.33-40.56), haemodialysis (IRR 8.59; 95% CI 1.82-40.53), invasive procedures (IRR 5.66; 95% CI 2.11-15.16), and β-lactam/β-lactamase inhibitor combinations (IRR 3.92; 95% CI 1.68-9.13) or third/fourth generation cephalosporin (IRR 2.75; 95% CI 1.06-7.11) exposure within 3 months before enrolment. Evidence of previous CRE colonization/infection was a major risk factor for carbapenem resistance among Enterobacterales BSI. Compared with uninfected patients, evidence of previous MDRO colonization/infection and healthcare exposure were important risk factors for CRE BSI. Targeted screening, infection prevention and antimicrobial stewardship should focus on these high-risk patients.

Temporal evolution of bacterial species and their antimicrobial resistance characteristics in wound infections of war-related injuries in Ukraine from 2014 to 2023

This study continues surveillance of antimicrobial resistance associated with combat injuries in Ukraine. To compare species composition, antibiotic resistance profiles, and emergence of new resistance genes between 2014-2020 and 2022-2023. This was a retrospective multi-centre microbiological survey in Ukrainian hospitals. Antibiotic susceptibility, whole-genome sequencing and multi-locus sequence typing were conducted on 154 organisms obtained from 125 casualties between 2022 and 2023. The data revealed a predominance of Gram-negative bacteria, particularly Acinetobacter baumannii (35.7%), Pseudomonas aeruginosa (14.9%) and Klebsiella pneumoniae (20.7%). High levels of carbapenem resistance were observed among A.baumannii {meropenem 72.2% [39/54, 95% confidence interval (CI) 58.4-83.5]; imipenem 66.7% (36/54, 95% CI 52.5-78.9)}, K.pneumoniae [meropenem 90.6% (29/32, 95% CI 75.0-98.0); imipenem 81.2% (26/32, 95% CI 63.6-92.8)] and P.aeruginosa [meropenem 47.8% (11/23, 95% CI 26.8-69.4); imipenem 60.8% (14/23, 95% CI 38.5-80.3)] strains. A.baumannii sequence type (ST)-78 and ST-400 were prevalent from 2014 to 2020, while five strains of ST-1077 were newly identified in 2022-2023. P. aeruginosa strains showed diversity across 16 STs, with ST-773 increasing in frequency and new STs emerging, but lacking carbapenemase genes. K.pneumoniae exhibited increased genetic diversity over time, with three STs from 2014 to 2020 and six new STs, including blaNDM-1, blaOXA-48 and blaKPC2 carriers, in 2022-2023. The prevalence of multi-drug-resistant isolates with STs associated with a high risk of global dissemination is increasing.

Molecular, Genetic, and Biochemical Characterization of OXA-484 Carbapenemase, a Difficult-to-Detect R214G Variant of OXA-181.

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.

Analysis of the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in a hospital in Hunan Province

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, β-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple β-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.

Incidence, antimicrobial resistance and mortality of Klebsiella pneumoniae bacteraemia in Shanghai, China, 2018–2022

Background Klebsiella pneumoniae (KP) accounts for high antimicrobial resistance and mortality rates of bloodstream infections (BSIs). Objectives To investigate incidence, antimicrobial resistance and risk factors for mortality of KP BSIs in East China. Methods A retrospective study of patients with KP BSIs was conducted in a tertiary care hospital from 2018 to 2022. Medical records of all hospitalised patients with KP BSIs were reviewed and analysed. The incidence, antimicrobial resistance and mortality of KP BSIs were evaluated. The Kaplan-Meier method was used to plot survival curves and logistic regression was used to analyse risk factors for crude 30-day mortality. Results A total of 379 inpatients with KP BSIs were enrolled. The incidence of patients with KP BSIs was fluctuating between 4.77 and 9.40 per 100,000 patient-days. The crude 30-day mortality rate of these patients was 26.39%. Of the 379 KPisolates, 197 (51.98%) were carbapenem-resistant (CR) and 252 (66.49%) were multidrug-resistant (MDR). All isolates showed the lowest resistance to tigecycline (13.77%) and polymyxin B (14.61%). Cases with MDR/CR isolates had significantly longer length of hospital stay, higher crude 30-day mortality and medical costs than non-MDR/non-CR isolates. Age, CR phenotype, paracentesis, indwelling central venous catheter (CVC), use of carbapenems, tetracyclines, polymyxins B, and irrational empiric treatment were independently associated with crude 30-day mortality. Conclusion MDR/CR KP BSIs are associated with increased mortality, healthcare costs and prolonged hospitalisation. Patients with advanced age, CR phenotype, paracentesis, CVC, exposure to some antibiotics, and irrational empirical antibiotic treatment are at higher mortality risk.