Carbapenem-resistant Isolates Research Articles

Extended-Spectrum Beta-Lactamase- and Plasmidic AmpC-Producing Enterobacterales among the Faecal Samples in the Bulgarian Community.

The aim of the present work was to genetically characterise cefotaxime-resistant enterobacteria isolated from community carriers in Bulgaria. In total, 717 faecal samples from children and adults in five medical centres in Sofia, Pleven and Burgas were examined. Antimicrobial susceptibility was evaluated by the disk diffusion method. blaESBL or plasmidic AmpC (pAmpC) genes were detected by PCR and sequencing. MLST and ERIC-PCR were used to detect clonal relatedness. Among the faecal samples, 140 cefotaxime-resistant enterobacteria were found. The most frequently detected species was Escherichia coli (77.9%, 109/140 samples), followed by Klebsiella pneumoniae (7.9%, 11/140). Among the isolates, blaCTX-M-15 (37.1%) was predominant, followed by blaCTX-M-3 (19.2%), blaCTX-M-14 (10%), and blaCTX-M-27 (4.3 %). Genes encoding pAmpC were observed in 11.4% (blaDHA-1, 16/140) and in 1.4% (blaCMY-2, 2/140). The frequency of ESBL and pAmpC producers among the subjects was 14.6% and 2.5%, respectively. No carbapenem-resistant isolates were found. Four main clonal complexes (CC131, CC10, CC38, and CC155) were detected among E. coli isolates. The most common type was ST131, phylogroup B2 (16.5%). The increased frequency of ESBL- and pAmpC-producing enterobacteria in the community is a prerequisite for treatment failures of the associated infections and a good background for further studies.

Evaluation of the Reveal® AST (SPECIFIC) for Antimicrobial Susceptibility Testing from Positive Blood Culture Spiked with Carbapenem-Resistant Isolates.

As bloodstream infections and associated septic shock are common causes of mortality in hospitals, rapid antibiotic susceptibility testing (AST) performed directly on positive blood cultures is needed to implement an efficient therapy in clinical settings. We evaluated the Reveal® rapid AST system on a collection of 197 fully characterized carbapenem-resistant Enterobacterales, including 177 carbapenemase producers (CPE) spiked in blood culture bottles. The clinical categorization based on the Minimal Inhibitory Concentration (MIC) determination of eighteen antimicrobial molecules was compared to the clinical categorization based on the disk diffusion assay as a reference. The Reveal AST system provided results within a mean time to result of 5 h. Overall, the categorical agreement (CA) between the two techniques was 94.1%. The rates of very major errors (VMEs), major errors (MEs) and minor errors (mEs) were 3.8%, 3.7% and 5.6%, respectively. Imipenem was the antimicrobial with the lowest CA rate (78.7%), with rates of 15% VMEs and 10.7% MEs, but the performances were better when considering only the non-CPE category (CA of 89%). On this resistant collection of Enterobacterales with numerous acquired β-lactamases, the Specific Reveal assay proved to be useful for a rapid determination of AST compatible with a quick adaptation of the patient's antimicrobial treatment.

Tigecycline Resistant Pattern among Carbapenem Resistant Gram-negative Bacilli: Prospective Cross-sectional Study

Introduction: Tigecycline is a unique tetracycline class of semi-synthetic, last-line broad spectrum antibiotic against multi-drug-resistant bacteria. However, recently, resistance to this antibiotic is on the rise. Aims: This study was conducted to determine the prevalence of tigecycline resistance amongst carbapenem-resistant Gram-negative bacilli (GNB) isolated from clinical samples (pus and sputum) as well as to evaluate their antimicrobial susceptibility pattern. Study design: Prospective cross sectional Place and Duration of Study: Department of Microbiology at Sanjay Gandhi Post Graduate Institute of Medical Sciences Lucknow, between January 2023 and December 2023. Methodology: Identification of GNB grown on culture was done by conventional biochemical tests and later validated by MALDI-TOF MS. The antimicrobial sensitivity testing of isolates was done using the E-test, and disk diffusion method. Minimum inhibitory concentration determination was done by Broth micro-dilution (BMD) method. Results: Amongst 8326 pus and respiratory samples, GNBs were recovered from 63.15% (5258/8326). Of 5258 GNB isolates, 50.74% (2668) were carbapenem-resistant, while 7.85% (413) demonstrated resistance to both tigecycline and carbapenem. Common isolates in this group were Klebsiella pneumoniae (37.04%), Acinetobacter spp. (25.18%), Enterobacter spp. (14.28%) and Escherichia coli (12.59%). BMD results demonstrated highest activity of tigecycline against carbapenem-resistant E. coli, followed by Citrobacter and Enterobacter. It works against resistant strains of Acinetobacter baumannii and K. pneumoniae as well, but in higher concentrations. Conclusion: High tigecycline resistance (one of the last-resort drugs) among carbapenem resistant GNB isolates is a matter of clinical concern, leaving physicians with limited options for treatment of such infections. Proper adherence to the policies of antimicrobial stewardship programs can reduce the emergence of resistance.

Surveillance of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Clinical Isolates in a Tertiary Care Hospital.

Antibiotic resistance among Gram-negative bacterial isolates is increasingly observed. With the emergence of carbapenem-resistant and pan-resistant pathogens, treating these resistant infections is becoming more challenging due to the limited number of effective drugs. There is a desperate need for the discovery of new antibiotics with novel mechanisms of action. Cefiderocol is one such novel antibiotic with a unique siderophore-based mechanism of action, which has been recently approved for clinical use against drug-resistant pathogens. The present study aims to identify the in vitro activity of cefiderocol against major carbapenem-resistant clinical isolates, including those resistant to colistin. One hundred and one carbapenem-resistant clinical isolates were included in the study. Identification and antibiotic susceptibility testing were performed using the automated VITEK® 2 Compact (bioMérieux SA, Marcy-l'Étoile, France) identification and susceptibility testing system, except for colistin and cefiderocol. Colistin resistance in Enterobacterales and Pseudomonas aeruginosa was assessed using the agar dilution minimum inhibitory concentration method, while for Acinetobacter baumannii, broth microdilution method was employed. Cefiderocol susceptibility testing was conducted using the Kirby-Bauer disc diffusion method with 30 µg discs on standard Mueller-Hinton agar plates. For selected isolates, cefiderocol minimum inhibitory concentration detection was performed using broth microdilution with iron-depleted cation-adjusted Mueller-Hinton broth. Of the total 101 isolates, the majority (75, 74.25%) were Enterobacterales which includedKlebsiella pneumonia (42, 41.58%) and Escherichia coli (33, 32.67%), followed by Pseudomonas aeruginosa (13, 12.87%) and Acinetobacter baumannii (10, 9.9%). Only three (2.97%)of the isolates were Stenotrophomonas maltophilia. Most of the isolates were susceptible to cefiderocol, with only four (3.96%) isolates showing resistance. Colistin resistance was observed in six (6.12%) of the isolates. There was a good correlation between disc diffusion testing and broth microdilution testing for the detection of cefiderocol-resistant isolates. No cross-resistance with colistin was observed, as all colistin-resistant isolates were uniformly susceptible to cefiderocol Conclusion: Cefiderocol is highly effective with good in vitro activity against the majority of carbapenem-resistant pathogens. While some isolates do show resistance, it is relatively uncommon. Given its safety profile compared to colistin, cefiderocol can serve as an alternative to colistin to treat carbapenem-resistant infections and it may be considered even for the management of colistin-resistant cases. Disc diffusion testing is a reliable method for identifying cefiderocol-resistant isolates in routine clinical and diagnostic laboratories, especially in resource-limited settings.

Prevalence and distribution of carbapenem-resistant Enterobacterales in companion animals: A nationwide study in the United States using commercial laboratory data.

Carbapenem-resistant Enterobacterales (CRE) are a concern in both human and animal medicine globally. Despite extensive research in humans, limited data exist on CRE in companion animals, with a lack of nationwide prevalence estimates. To assess the occurrence and trends of CRE in cats and dogs across the United States by analyzing 4 years of commercial antimicrobial susceptibility testing (AST) data. Between 2019 and 2022, 477 426 ASTs were conducted on Enterobacterales isolates against imipenem. Isolates were derived from 379 598 dogs and 97 828 cats. Animal origin was not disclosed. In this retrospective study, antimicrobial susceptibility test data from IDEXX Laboratories were analyzed. Analysis included resistance estimations to imipenem stratified by sampling site, an assessment of resistance patterns over time and location, and the application of space-time cluster analysis to identify potential outbreaks. Antibiograms were produced for carbapenem-resistant isolates. Susceptibility to imipenem was high, at 98.86%. Temporal analysis indicated stability in susceptibility, with an unexplained reduction in susceptible isolates in June 2019. Spatial analysis identified 2 high-risk clusters along the Western Coast (relative risk [RR]: 23.26; P < .001) and in Texas (RR: 10.72; P < .001) in that month. Three other clusters were found, in Missouri (RR: 39.55; P = .038), Florida (RR: 4.53; P < .001), and New York (RR: 9.20; P < .001). CRE are present at a low prevalence in dogs and cats across the United States. Variations in prevalence across patient-level and environmental factors highlight the need for tailored stewardship programs.

Occurrence of blaNDM-1, blaNDM-5, blaNDM-7, and blaKPC-2 genes in clinical isolates of enterobacterales with high genetic variability, from colonization and infection in patients with or without COVID-19, from a hospital in Brazil.

This study aimed to investigate the presence of beta-lactams resistance genes and the clonal relationship of clinical isolates of Enterobacterales obtained from patients with and without COVID-19, in a hospital in northeastern Brazil. The study analyzed 45 carbapenem-resistant clinical isolates using enterobacterial repetitive intergenic consensus (ERIC-PCR), PCR, and amplicon sequencing to detect resistance genes (blaKPC, blaGES, blaNDM, blaVIM, and blaIMP). The main species were Klebsiella pneumoniae, Serratia marcescens, and Proteus mirabilis. Detected genes included blaNDM (46.66%), blaKPC (35.55%), and both (17.79%). ERIC-PCR showed multiclonal dissemination and high genetic variability. The main resistance gene was blaNDM, including blaNDM-5 and blaNDM-7. The presence of Enterobacterales carrying blaKPC and blaNDM in this study, particularly K. pneumoniae, in infections and colonizations of patients with COVID-19 and non-COVID-19, highlights genetic variability and resistance to carbapenems observed in multiple species of this order.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

Synergistic Activity of Cefiderocol in Combination with Avibactam, Sulbactam or Tazobactam against Carbapenem-Resistant Gram-Negative Bacteria.

We investigated the activity of cefiderocol/β-lactamase inhibitor combinations against clinical strains with different susceptibility profiles to cefiderocol to explore the potentiality of antibiotic combinations as a strategy to contain the major public health problem of multidrug-resistant (MDR) pathogens. Specifically, we evaluated the synergistic activity of cefiderocol with avibactam, sulbactam, or tazobactam on three of the most "Critical Priority" group of MDR bacteria (carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii). Clinical isolates were genomically characterized by Illumina iSeq 100. The synergy test was conducted with time-kill curve assays. Specifically, cefiderocol/avibactam, /sulbactam, or /tazobactam combinations were analyzed. Synergism was assigned if bacterial grow reduction reached 2 log10 CFU/mL. We reported the high antimicrobial activity of the cefiderocol/sulbactam combination against carbapenem-resistant Enterobacterales, P. aeruginosa, and A. baumannii; of the cefiderocol/avibactam combination against carbapenem-resistant Enterobacterales; and of the cefiderocol/tazobactam combination against carbapenem-resistant Enterobacterales and P. aeruginosa. Our results demonstrate that all β-lactamase inhibitors (BLIs) tested are able to enhance cefiderocol antimicrobial activity, also against cefiderocol-resistant isolates. The cefiderocol/sulbactam combination emerges as the most promising combination, proving to highly enhance cefiderocol activity in all the analyzed carbapenem-resistant Gram-negative isolates, whereas the Cefiderocol/tazobactam combination resulted in being active only against carbapenem-resistant Enterobacterales and P. aeruginosa, and cefiderocol/avibactam was only active against carbapenem-resistant Enterobacterales.

Molecular detection of extended-spectrum β-lactamase- and carbapenemase-producing Klebsiella pneumoniae isolates in southwest Iran.

The global emergence of carbapenem-resistant Klebsiella pneumoniae is considered a significant contemporary concern., as carbapenems are the last resort for treating infections caused by multidrug-resistant Gram-negative bacteria. This study aimed to investigate the carbapenem-resistance genes in extended-spectrum β-lactamase producing K. pneumoniae isolates. Seventy-five non-duplicate clinical K. pneumoniae strains were isolated from urine, blood, sputum, and wound samples. Antimicrobial susceptibility tests for 12 different antibiotics were performed using the disk diffusion method, followed by determining minimum inhibitory concentrations of imipenem and meropenem. Phenotypic detection of extended-spectrum β-lactamase and carbapenemase enzymes was performed by double-disc synergy test and modified Hodge test, respectively. PCR assay further investigated resistant isolates for extended-spectrum β-lactamase and carbapenemase-encoding genes. The highest and lowest resistance rates were observed against ampicillin (93.3%) and tigecycline (9.3%). According to phenotypic tests, 46.7% of isolates were positive for extended-spectrum β-lactamase enzymes and 52.8% for carbapenemase. A total of 11 isolates contained carbapenemase genes, with blaOXA-48 (19.4%; 7/36) being the predominant gene, followed by blaNDM (8.3%; 3/36). The study's findings reveal the alarming prevalence of beta-lactamase enzymes in K. pneumoniae strains. Early detection of carbapenem-resistant isolates and effective infection control measures are necessary to minimise further spread, as carbapenem resistance has become a public health concern.

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

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

Biological studies of the activity of Manuka honey against Carbapenem-resistant Enterobacterales (CRE) bacteria.

To evaluate the potency of Manuka honey UMF +15 against Carbapenem-resistant Enterobacterales (CRE). Bacterial resistance is a worldwide problem that is increasing year by year, especially Carbapenem resistance. Alternatives to antibiotics are needed to both reduce costs, and to reduce the spread of antibiotic resistance, with the ultimate goal of saving lives. The efficacy of Manuka honey UMF +15 was tested by 2 methods; Well diffusion assay and minimum bactericidal concentration (MBC) against twenty Carbapenem-resistant isolates which collected from Makkah city hospitals during three months of study from 1st of September 2023 up to 1st of December 2023. The growth of all isolates of Carbapenem-resistant Enterobacterales (CRE) was severely inhibited by low concentrations of Manuka honey, affecting 25% of isolates at 15% and 75% of isolates at 18% of Manuka honey. In addition, using the honey at different concentrations in a well diffusion assay resulted, as expected, in a variable zone diameter, ranging from large zones(14mm) to small zones (2mm) according to the concentration of the honey. This study shows the remarkable antibacterial activity of Manuka honey and suggests that this natural remedy might be used in the future as an alternative treatment option against Carbapenem-resistant Enterobacterales (CRE); however, further clinical trials should be performed to corroborate our initial findings.

Assessment of carbapenem resistance and carbapenemase genes in wastewater from cattle slaughterhouses: Implications for environmental antibiotic resistance surveillance

The objectives of the study were to determine the prevalence of carbapenem-resistant Gram-negative bacteria and assess the potential risks associated with cattle slaughterhouse wastewater. A total of 270 wastewater samples were collected from 10 different cattle slaughterhouses for microbiological analysis. Conventional culture methods were employed, followed by disc diffusion, the Modified Carbapenem Inactivation Method (mCIM), and the Modified Hodge Test (MHT) to identify carbapenem resistance. The Vitek® 2 compact system was used for species identification and antibiotic susceptibility profiling. Conventional and quantitative PCR (qPCR) were performed to detect specific carbapenemase genes (blaKPC, blaNDM, and blaOXA-48). Among the collected 72 carbapenem-resistant isolates, one Pseudomonas fluorescens, one Aeromonas hydrophila, and two Aeromonas sobria exhibited resistance to meropenem. Additionally, six P. fluorescens and two A. hydrophila isolates demonstrated intermediate resistance to meropenem. Furthermore, five carbapenem-resistant isolates were identified as Stenotrophomonas maltophilia, known to be inherently resistant to most antibiotics. Ten different antibiotics were evaluated in the antibiotic resistance panel and all Aeromonas isolates were found to be resistant to cefazolin and one A. hydrophila was detected as multi-drug resistant. The revealed data indicates that slaughterhouse wastewater can serve as a reservoir for antibiotic-resistant opportunistic pathogens. However, it may not pose a substantial risk for the distribution of carbapenemases, thereby mitigating concerns related to potential public health and environmental hazards associated with this aspect of slaughterhouse operations. This study contributes to understanding of antibiotic resistance in livestock-related environments and underscores the importance of continued monitoring and surveillance.

Genome-wide sRNA and mRNA transcriptomic profiling insights into carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii (AB) is rising as a human pathogen of critical priority worldwide as it is the leading cause of opportunistic infections in healthcare settings and carbapenem-resistant AB is listed as a "super bacterium" or "priority pathogen for drug resistance" by the World Health Organization. Clinical isolates of A. baumannii were collected and tested for antimicrobial susceptibility. Among them, carbapenem-resistant and carbapenem-sensitive A. baumannii were subjected to prokaryotic transcriptome sequencing. The change of sRNA and mRNA expression was analyzed by bioinformatics and validated by quantitative reverse transcription-PCR. A total of 687 clinical isolates were collected, of which 336 strains of A. baumannii were resistant to carbapenem. Five hundred and six differentially expressed genes and nineteen differentially expressed sRNA candidates were discovered through transcriptomic profile analysis between carbapenem-resistant isolates and carbapenem-sensitive isolates. Possible binding sites were predicted through software for sRNA21 and adeK, sRNA27 and pgaC, sRNA29 and adeB, sRNA36 and katG, indicating a possible targeting relationship. A negative correlation was shown between sRNA21 and adeK (r = -0.581, P = 0.007), sRNA27 and pgaC (r = -0.612, P = 0.004), sRNA29 and adeB (r = -0.516, P = 0.020). This study preliminarily screened differentially expressed mRNA and sRNA in carbapenem-resistant A. baumannii, and explored possible targeting relationships, which will help further reveal the resistance mechanism and provide a theoretical basis for the development of drugs targeting sRNA for the prevention and treatment of carbapenem-resistant A. baumannii infection.

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.

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.

Emergence of blaNDM-5 and blaOXA-232 Positive Colistin- and Carbapenem-Resistant Klebsiella pneumoniae in a Bulgarian Hospital.

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.

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.

Evaluation of the in vitro activity of ampicillin-sulbactam and cefoperazone-sulbactam against A. Baumannii by the broth disk elution test.

To evaluate the in vitro activity of ampicillin-sulbactam and cefoperazone-sulbactam against A. baumannii using the broth disk elution testing, a total of 150 A. baumannii isolates were collected from across China between January 2019 and January 2021, including 51 carbapenem-susceptible and 99 carbapenem-resistant isolates. Broth disk elution (BDE) and the broth microdilution (BMD) method were performed for all strains. The concentration range of the BDE was 10/10 µg/mL, 20/20 µg/mL, and 30/30 µg/mL for ampicillin-sulbactam, and 37.5/15 µg/mL, 75/30 µg/mL, 112.5/45 µg/mL, and 150/60 µg/mL for cefoperazone-sulbactam, respectively. Compared with BMD, the BDE results of ampicillin-sulbactam and cefoperazone-sulbactam showed a categorical agreement of 83.3% (125/150) and 95.3% (143/150), with minor errors of 16.7% (25/150) and 4.7% (7/150), respectively. No major error or very major errors were detected. The sensitivity differences by BDE of carbapenem-resistant A. baumannii (CRAb)to different concentrations of ampicillin-sulbactam showed statistically significant (p < 0.017), while those to cefoperazone-sulbactam at 37.5/15 µg/mL, 75/30 µg/mL, and 112.5/45 µg/mL were significant (p < 0.008). However, no significant difference in sensitivity was observed between 112.5/45 µg/mL and 150/60 µg/mL (p > 0.008). In conclusion, the BDE is a reliable and convenient method to detect the in vitro activity of cefoperazone-sulbactam against A. baumannii, and the results could serve as a clinical reference value when deciding whether or not to use high-dose sulbactam for the treatment of A. baumannii infections.

Efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli.

Carbapenem-resistant Enterobacterales (CRE) have become a major public health problem worldwide. The aim of this study was to investigate efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates. Susceptibility of imipenem, meropenem, ertapenem, ceftazidime/avibactam and plazomicin was investigated by broth-microdilution method. Major carbapenemases NDM, VIM, IMP, KPC, OXA-48 as well as other β-lactamases namely, TEM, SHV, OXA-1-like, CTX-M, ACC, FOX, MOX, DHA, CIT, EBC, VEB, GES, PER were investigated by PCR. A total of 120 carbapenem-resistant isolates (60 E. coli and 60 K. pneumoniae) were included in this study and blaOXA-48-like was found in 78.33%, blaNDM in 26.66%, blaKPC in 7.5%, blaIMP in 5.83%, and blaVIM in 5%. Among 94 isolates with the blaOXA-48-like gene, 22.3% were resistant to ceftazidime/avibactam and 51.1% were resistant to plazomicin. Of 32 isolates with blaNDM, 31 (96.9%) were resistant to ceftazidime/avibactam and 30 (93.75%) were resistant to plazomicin, and both antibiotics had limited effects against blaNDM carriers (P < 0.001). Of the 12 isolates with blaNDM+OXA-48 combination, 11 (91.7%) were resistant to ceftazidime/avibactam and plazomicin. The effect of both antibiotics was significantly lower in strains with blaNDM+OXA-48 combination (P < 0.005).The most common carbapenemase genes in this study were blaOXA-48-like and blaNDM. Ceftazidime/avibactam demonstrated a good efficacy among OXA-48 producing K. pneumoniae and E. coli, however, plazomicin had a significantly lower antibacterial effect in our study. Both antimicrobial agents should be considered as an option by evaluating combined susceptibility results and gene patterns obtained by regional and global molecular data in the treatment of CRE infections.

Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-β-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against β-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.