Molecular characterization of hypermucoviscous carbapenemase-encoding Klebsiella pneumoniae isolates from an Egyptian hospital.

This study aimed to screen antibiotic resistance and virulence genes in carbapenem-resistant hypermucoviscous Klebsiella pneumoniae isolates from an Egyptian hospital. Among 38 previously confirmed carbapenem-nonsusceptible K. pneumoniae isolates, a string test identified three isolates as positive for hypermucoviscosity. Phenotypic characterization and molecular detection of carbapenemase- and virulence-encoding genes were performed. PCR-based multilocus sequence typing and phylogenetics were used to determine the clonality and global epidemiology of the strains. The coexistence of virulence and resistance genes in the isolates was analyzed statistically using a chi-square test. Three isolates showed the presence of carbapenemase-encoding genes (blaNDM, blaVIM, and blaIMP), adhesion genes (fim-H-1 and mrkD), and siderophore genes (entB); the isolates belonged to sequence types (STs) 101, 1310, and 1626. The relatedness between these sequence types and the sequence types of globally detected hypermucoviscous K. pneumoniae that also harbor carbapenemases was determined. Our analysis showed that the resistance and virulence profiles were not homogenous. Phylogenetically, different clones clustered together. There was no significant association between the presence of resistance and virulence genes in the isolates. There is a need for periodic surveillance of the healthcare settings in Egypt and globally to understand the true epidemiology of carbapenem-resistant, hypermucoviscous K. pneumoniae.

Epidemiology and clinical significance of carbapenemases in Australia: a narrative review.

Carbapenemase-producing gram-negative bacteria (CP-GNB) infections threaten public health with high mortality, morbidity and treatment costs. Although frequencies remain low in Australia (total number of CP-GNB infections reported was 907 in 2022), blaIMP-4 has established low levels of endemicity in many states. Imipenemase metallo-ß-lactamase types alone accounted for more than half of all carbapenemases in carbapenemase-producing Enterobacterales isolates in Australia, particularly in Enterobacter cloacae complex. New Delhi metallo-ß-lactamase constitutes almost 25% of all carbapenemases in Australia and was identified predominantly in Escherichia coli. The OXA-48-like carbapenemases include almost 10% of all carbapenemases and are mainly seen in Klebsiella pneumoniae and E. coli. Although K. pneumoniae carbapenemase-type carbapenemases are rare in Australia, some local outbreaks have occurred. Most carbapenem-resistant (CR) Pseudomonas aeruginosa strains in Australia do not produce carbapenemases. Finally, OXA-23-like carbapenemases are overwhelmingly positive in CR-Acinetobacter baumannii strains in Australia. Treatment of CR-GNB infections challenges physicians. Of 10 new antibiotics active against at least some CR-GNB infections that are approved by the US Food and Drug Administration, just three are approved for use in Australia. In this context, there is still an unmet need for novel antibacterials that can be used for the treatment of CR-GNB infections in Australia, as well as a pressing requirement for new mechanisms to 'de-link' antibiotic sales from their availability. In this narrative review, we aim to overview the epidemiology and clinical significance of carbapenem resistance in Australia as it pertains to Enterobacterales, P. aeruginosa and A. baumannii.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Reliability of a Screening Method Using Antibiotic Disks to Detect Carbapenemases in Glucose-Nonfermenting Gram-Negative Microorganisms From Clinical Samples of a Regional Hospital in Southeastern Spain.

BACKGROUND: Infections by glucose-nonfermenting gram-negative bacilli (NFGNB) pose a major public health problem due to multiresistance to beta-lactam antibiotics, especially plasmid-borne carbapenemases. Their detection by microbiology laboratories is challenging, and there is a need for easy-to-use and reliable diagnostic techniques. Our objective was to evaluate an in-house screening method to presumptively detect carbapenemases in NFGNB in a simple and clinically useful manner. METHODS: The study included 175 NFGNB isolates from urinary, respiratory, and rectal samples. In a triple assay, isolates were incubated at 37°C for 24 h on three solid-culture media: MacConkey II Agar, 5% Sheep Blood Columbia Agar and Mueller Hinton II Agar; meropenem (MEM) and cefepime (FEP) disks were employed for screening. Studies were then performed on the inhibition halo diameter, scanning effects, and the appearance of mutant colonies, which were compared with those observed using the colorimetric Neo-Rapid CARB Kit and immunochromatography (NG5-Test Carba and K-Set for OXA-23). Receiver operating characteristic curves were constructed for these data. RESULTS: Carbapenemases were expressed by 79/175 (45.1%): 19 Pseudomonas aeruginosa and 60 Acinetobacter baumannii. Optimal inhibition halo diameter cutoffs to detect this resistance on 5% sheep blood agar were as follows: 6 mm (MEM) and 6.5 mm (FEP) for P. aeruginosa (in the absence of scanning effects and mutations) and 10.5 mm (MEM) and 16 mm (FEP) for A. baumannii (even in the presence of scanning effects). CONCLUSION: The combined utilization of MEM and FEP antibiotic disks in 5% sheep blood agar, measuring their inhibition haloes, offers an effective method to predict the presence of carbapenemases as resistance mechanism in P. aeruginosa and A. baumannii.

Emergence of OXA-48-like producing Citrobacter species, Germany, 2011 to 2022.

BackgroundCarbapenemase-producing Enterobacterales are a public health threat worldwide and OXA-48 is the most prevalent carbapenemase in Germany and western Europe. However, the molecular epidemiology of OXA-48 in species other than Escherichia coli and Klebsiella pneumoniae remains poorly understood.AimTo analyse the molecular epidemiology of OXA-48 and OXA-48-like carbapenemases in Citrobacter species (spp.) in Germany between 2011 and 2022.MethodsData of 26,822 Enterobacterales isolates sent to the National Reference Centre (NRC) for Gram-negative bacteria were evaluated. Ninety-one Citrobacter isolates from 40 German hospitals harbouring bla OXA-48/OXA-48­like were analysed by whole genome sequencing and conjugation experiments.ResultsThe frequency of OXA-48 in Citrobacter freundii (CF) has increased steadily since 2011 and is now the most prevalent carbapenemase in this species in Germany. Among 91 in-depth analysed Citrobacter spp. isolates, CF (n = 73) and C. koseri (n = 8) were the most common species and OXA-48 was the most common variant (n = 77), followed by OXA-162 (n = 11) and OXA­181 (n = 3). Forty percent of the isolates belonged to only two sequence types (ST19 and ST22), while most other STs were singletons. The plasmids harbouring bla OXA­48 and bla OXA-162 belonged to the plasmid types IncL (n = 85) or IncF (n = 3), and plasmids harbouring bla OXA­181 to IncX3 (n = 3). Three IncL plasmid clusters (57/85 IncL plasmids) were identified, which were highly transferable in contrast to sporadic plasmids.ConclusionIn CF in Germany, OXA-48 is the predominant carbapenemase. Dissemination is likely due to distinct highly transmissible plasmids harbouring bla OXA­48 or bla OXA-48-like and the spread of the high-risk clonal lineages ST19 and ST22.

Biodiversity of carbapenem-resistant bacteria in clinical samples from the Southwest Amazon region (Rondônia/Brazil).

Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (blaKPC-like), B (blaNDM-like, blaSPM-like or blaVIM-like) and D (blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like or blaOXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

Rapid diagnostic of multidrug-resistant sepsis pathogens directly from blood culture bottles using MALDI-TOF and the EUCAST RAST.

In this study, rapid diagnostic of multidrug-resistant (MDR) sepsis pathogens, directly from positive blood culture (BC) bottles, was evaluated by combining MALDI-TOF and the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST). Carbapenemase production in Escherichia coli and Klebsiella pneumoniae isolates was also evaluated by RAST. From 171 positive BC bottles analyzed, 79 (46 %) MDR species, including E. coli (4/34, 12 %), K. pneumoniae (33/48, 69 %), Pseudomonas aeruginosa (12/12, 100 %), Acinetobacter baumannii (15/15, 100 %), and Staphylococcus aureus (14/37, 38 %) displaying resistance to beta-lactams, fluoroquinolones, aminoglycosides, and/or trimethoprim/sulphamethoxazole, were identified. In this regard, turnaround time of direct MALDI-TOF identification and RAST was < 7 h, which was significantly (p< 0.05) lower than our routine method. Carbapenemase detection by RAST displayed 100% sensitivity and 88.7 % specificity at 8 h. This protocol could offer advantages for the treatment and clinical outcomes of septic patients, improving the rapid diagnostic of sepsis by MDR pathogens.

Coexistence of blaNDM-5, blaCTX-M-15, blaOXA-232, blaSHV-182 genes in multidrug-resistant K. pneumoniae ST437-carrying OmpK36 and OmpK37 porin mutations: First report in Italy.

OBJECTIVES: K. pneumoniae is a common cause of severe hospital-acquired infections. In the present study, we have characterised the whole-genome of two K. pneumoniae ST437 belonging to the clonal complex CC258. METHODS: The whole-genome sequencing was performed by MiSeq Illumina, with a 2 × 300bp paired-end run. ResFinder 4.4.2 was used to detect acquired antimicrobial resistance genes (ARGs) and chromosomal mutations. Mobile genetic elements (plasmids and ISs) were identified by MobileElementFinder v1.0.3. The genome was also assigned to ST using MLST 2.0.9. Virulence factors were detected using the Virulence Factor Database (VFDB). RESULTS: K. pneumoniae KPNAQ_1/23 and KPNAQ_2/23 strains, isolated from urine samples of hospitalised patients, showed resistance to most antibiotics, including ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam combinations. Both strains were susceptible only to cefiderocol. Multiple mechanisms of resistance were identified. Resistance to ß-lactams was due to the presence of NDM-5, OXA-232, CTX-M-15, SHV-182 ß-lactamases, and OmpK36 and OmpK37 porin mutations. Resistance to fluoroquinolones was mediated by chromosomal mutations in acrR, oqxAB efflux pumps, and the bifunctional gene aac(6')-Ib-cr. CONCLUSION: The presence of different virulence genes makes these KPNAQ_1/23 and KPNAQ_2/23 high-risk clones.

Reduce susceptibility to cefiderocol in gram negative bacteria in children: Is hope already lost before it's even arrived?

BACKGROUND: In last years the diffusion of carbapenem resistance in Gram-negative bacteria (CR-GNB) is increasing worldwide, mainly due to the expression of carbapenemases. Cefiderocol has molecular characteristics that ideally confers activity against all CR-GNB, but resistant strains have already been identified. We describe cefiderocol susceptibility profile among multi-drug resistant Gram-negative isolated from pediatric patients. METHODS: Prospective, single pediatric center study, 1st January 2020-15th June 2023. All GNB carbapenemases producers or phenotypically carbapenem-resistant isolated in the study period were tested for cefiderocol susceptibility. Clinical and microbiological data were collected. A descriptive analysis was performed, comparing the groups of cefiderocol-resistant vs. cefiderocol-susceptible Enterobacterales and non-fermenting Gram-negative bacteria (NF-GNB). RESULTS: Forty-seven GNB were tested for cefiderocol susceptibility; 38% were cefiderocol-resistant: 16/30 (52%) among Enterobacterales and 2/17 (12%) among NF-GNB. None of the patients were previously exposed to cefiderocol. Looking at Enterobacterales, resistance to ceftazidime/avibactam was higher among cefiderocol-resistant vs. cefiderocol-susceptible strains (62% vs 36%, respectively), as MBL expression (67% vs. 36%, respectively). Too few NF-GNB were cefiderocol-resistance to draw any conclusion. No difference in ICU admission and mortality was identified comparing cefiderocol-resistant vs. susceptible strains. Patients colonized/infected by cefiderocol-resistant strains had been previously hospitalized more frequently. CONCLUSION: In our cohort cefiderocol resistance was mostly registered among Enterobacterales, and especially among MBL producers' strains (that were alongside resistant to ceftazidime/avibactam). This could be explained by the known possible cross resistance mechanism among ceftazidime/avibactam and cefiderocol. Also, correlation of cefiderocol-resistance with previous hospitalization could be associated with horizontal resistance transmission. Looking at our data, we believe that cefiderocol should be use cautiously, especially empirically and in monotherapy, due to the high resistance rate.

Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strains have become a global threat due to their remarkable capability to survive and disseminate successfully by the acquisition of resistance genes. As a result, the treatment strategies have been severely compromised. Due to the insufficient available data regarding P. aeruginosa resistance from Pakistan, we aimed to investigate the resistance mechanisms of 249 P. aeruginosa strains by antimicrobial susceptibility testing, polymerase chain reaction for the detection of carbapenemases, aminoglycoside resistance genes, extended-spectrum beta-lactamases (ESBLs), sequence typing and plasmid typing. Furthermore, we tested silver nanoparticles (AgNPs) to evaluate their in vitro sensitivity against antimicrobial-resistant P. aeruginosa strains. We observed higher resistance against antimicrobials in the general surgery ward, general medicine ward and wound samples. Phenotypic carbapenemase-producer strains comprised 80.7% (201/249) with 89.0% (179/201) demonstrating genes encoding carbapenemases: blaNDM-1 (32.96%), blaOXA48 (37.43%), blaIMP (7.26%), blaVIM (5.03%), blaKPC-2 (1.12%), blaNDM-1/blaOXA48 (13.97%), blaOXA-48/blaVIM (1.68%) and blaVIM/blaIMP (0.56%). Aminoglycoside-modifying enzyme genes and 16S rRNA methylase variants were detected in 43.8% (109/249) strains: aac(6')-lb (12.8%), aac(3)-lla (12.0%), rmtB (21.1%), rmtC (11.0%), armA (12.8%), rmtD (4.6%), rmtF (6.4%), rmtB/aac(3)-lla (8.2%), rmtB/aac(6')-lla (7.3%) and rmtB/armA (3.6%). In total, 43.0% (77/179) of the strains coharbored carbapenemases and aminoglycoside resistance genes with 83.1% resistant to at least 1 agent in 3 or more classes and 16.9% resistant to every class of antimicrobials tested. Thirteen sequence types (STs) were identified: ST235, ST277, ST234, ST170, ST381, ST175, ST1455, ST1963, ST313, ST207, ST664, ST357 and ST348. Plasmid replicon types IncFI, IncFII, IncA/C, IncL/M, IncN, IncX, IncR and IncFIIK and MOB types F11, F12, H121, P131 and P3 were detected. Meropenem/AgNPs and Amikacin/AgNPs showed enhanced antibacterial activity. We reported the coexistence of carbapenemases and aminoglycoside resistance genes among carbapenem-resistant P. aeruginosa with diverse clonal lineages from Pakistan. Furthermore, we highlighted AgNP's potential role in handling future antimicrobial resistance concerns.

Rapid AST: Possibility of inferring resistance mechanisms with complex phenotypes / Pruebas rápidas de sensibilidad a los antimicrobianos: ¿es posible inferir mecanismos de resistencia con fenotipos complicados?

The new automated systems designed for rapid performance of AST have significantly reduced the response time for susceptibility testing of microorganisms causing bacteremia and sepsis. The Accelerate Pheno® system (AAC) is one such system. Our objective for this study was to determine whether the AAC system is capable of providing an accurate susceptibility profile to infer resistance mechanisms in different carbapenemase-producing isolates when compared to the MicroScan WalkAway System (MWS). Disk diffusion method was also performed on all isolates as a reference method. Additionally, we compared the results obtained with the routine AST production system. We selected 19 isolates from the cryobank of the Microbiology department, all of which were carbapenemase-producing gram-negative bacilli. AAC was able to identify and infer the resistance of a total of 10 isolates, with an EA and CA of 84.2% for meropenem and 88.2% and 64.7% for ertapenem EA and CA, respectively. If we consider the disk diffusion technique, the CA was 57.9% and 76.5% for meropenem and ertapenem. However, in the presence of carbapenemases, AAC was not able to provide adequate MICs or infer the resistance mechanisms of the isolates accurately. Further studies with a larger number of isolates, including the new antibiotics ceftolozane/tazobactam and ceftazidime/avibactam, are needed for a more comprehensive comparison. (AU)

Los nuevos sistemas automatizados diseñados para la realización rápida de antibiogramas han reducido significativamente el tiempo de respuesta para las pruebas de susceptibilidad de los microorganismos causantes de bacteriemia y sepsis. El sistema Accelerate Pheno® (AAC) es uno de ellos. Nuestro objetivo para este estudio era determinar si el sistema AAC es capaz de proporcionar un perfil de sensibilidad preciso para inferir mecanismos de resistencia en diferentes aislados productores de carbapenemasas en comparación con el sistema MicroScan WalkAway (MWS). El método de disco difusión fue incluido también en todos los aislados como método de referencia. Además, comparamos los resultados obtenidos con el sistema rutinario de producción de antibiogramas rápidos. Seleccionamos 19 aislados del criobanco del departamento de Microbiología, todos ellos bacilos gramnegativos productores de carbapenemasas. AAC fue capaz de identificar e inferir la resistencia de un total de 10 aislados, con una EA y CA del 84,2% para el meropenem y del 88,2% y 64,7% para la EA y CA del ertapenem, respectivamente. Si consideramos la técnica de disco difusión, la CA fue de un 57.9% y de un 76.5% para meropenem y ertapenem. Sin embargo, en presencia de carbapenemasas, AAC no fue capaz de proporcionar CMIs adecuadas ni de inferir con precisión los mecanismos de resistencia de los aislados. Se necesitan más estudios con un mayor número de aislados incluyendo también los nuevos antibióticos ceftolozano/tazobactam y ceftazidima/avibactam para una comparación más exhaustiva. (AU)

Molecular characterization of carbapenem-resistance in Gram-negative isolates obtained from clinical samples at Jimma Medical Center, Ethiopia.

Background: In resource-constrained settings, limited antibiotic options make treating carbapenem-resistant bacterial infections difficult for healthcare providers. This study aimed to assess carbapenemase expression in Gram-negative bacteria isolated from clinical samples in Jimma, Ethiopia. Methods: A cross-sectional study was conducted to assess carbapenemase expression in Gram-negative bacteria isolated from patients attending Jimma Medical Center. Totally, 846 Gram-negative bacteria were isolated and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Phenotypic antibiotic resistance patterns were determined using the Kirby-Bauer disk diffusion method and Etest strips. Extended-spectrum ß-lactamase phenotype was determined using MAST disks, and carbapenemases were characterized using multiplex polymerase chain reactions (PCR). Results: Among the isolates, 19% (157/846) showed phenotypic resistance to carbapenem antibiotics. PCR analysis revealed that at least one carbapenemase gene was detected in 69% (107/155) of these strains. The most frequently detected acquired genes were blaNDM in 35% (37/107), blaVIM in 24% (26/107), and blaKPC42 in 13% (14/107) of the isolates. Coexistence of two or more acquired genes was observed in 31% (33/107) of the isolates. The most common coexisting acquired genes were blaNDM + blaOXA-23, detected in 24% (8/33) of these isolates. No carbapenemase-encoding genes could be detected in 31% (48/155) of carbapenem-resistant isolates, with P. aeruginosa accounting for 85% (41/48) thereof. Conclusion: This study revealed high and incremental rates of carbapenem-resistant bacteria in clinical samples with various carbapenemase-encoding genes. This imposes a severe challenge to effective patient care in the context of already limited treatment options against Gram-negative bacterial infections in resource-constrained settings.

Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Escherichia coli sequence type 410 with carbapenemases: a paradigm shift within E. coli toward multidrug resistance.

Escherichia coli sequence type ST410 is an emerging carbapenemase-producing multidrug-resistant (MDR) high-risk One-Health clone with the potential to significantly increase carbapenem resistance among E. coli. ST410 belongs to two clades (ST410-A and ST410-B) and three subclades (ST410-B1, ST410-B2, and ST410-B3). After a fimH switch between clades ST410-A and ST410-B1, ST410-B2 and ST410-B3 subclades showed a stepwise progression toward developing MDR. (i) ST410-B2 initially acquired fluoroquinolone resistance (via homologous recombination) in the 1980s. (ii) ST410-B2 then obtained CMY-2, CTX-M-15, and OXA-181 genes on different plasmid platforms during the 1990s. (iii) This was followed by the chromosomal integration of blaCMY-2, fstl YRIN insertion, and ompC/ompF mutations during the 2000s to create the ST410-B3 subclade. (iv) An IncF plasmid "replacement" scenario happened when ST410-B2 transformed into ST410-B3: F36:31:A4:B1 plasmids were replaced by F1:A1:B49 plasmids (both containing blaCTX-M-15) followed by blaNDM-5 incorporation during the 2010s. User-friendly cost-effective methods for the rapid identification of ST410 isolates and clades are needed because limited data are available about the frequencies and global distribution of ST410 clades. Basic mechanistic, evolutionary, surveillance, and clinical studies are urgently required to investigate the success of ST410 (including the ability to acquire successive MDR determinants). Such information will aid with management and prevention strategies to curb the spread of carbapenem-resistant E. coli. The medical community can ill afford to ignore the spread of a global E. coli clone with the potential to end the carbapenem era.

Different OXA-Carbapenemases in Genetically Unrelated Klebsiella pneumoniae Strains Isolated in a North Italian Hospital During Multidrug Resistance Screening.

Klebsiella pneumoniae is one of the main opportunistic pathogens that cause a broad spectrum of diseases with increasingly frequent acquisition of resistance to antibiotics, namely carbapenems. This study focused on the characterization of 23 OXA-48-like carbapenemase-producing K. pneumoniae isolates using phenotypic and molecular tests. Phenotypic determination of the presence of ß-lactamases was performed using the extended-spectrum beta-lactamase (ESBL) NP test, and phenotypic determination of the presence of carbapenemase was based on the Carba NP test. Antimicrobial susceptibility tests were performed to assess the resistance against carbapenems. Molecular characterization of ESBL genes and carbapenemase genes (blaOXA-48, blaKPC, blaVIM, and blaNDM) was performed using polymerase chain reaction (PCR) techniques. In addition, K. pneumoniae strains were analyzed for their relatedness using multilocus sequence typing PCR analysis based on the Institut Pasteur protocol, which produces allelic profiles that contain their evolutionary and geographic pattern. Following further Sanger sequencing of the blaOXA-48 genes, no genetic mutations were found. Some OXA-48-producing K. pneumoniae isolates coharbored blaKPC, blaNDM, and blaVIM genes, which encode other carbapenemases that can hydrolyze carbapenem antibiotics. The final part of the study focused on the characterization of the plasmid profiles of all isolates to better understand the spreading of the IncL/M blaOXA-48 plasmid gene. The plasmid profile also revealed other incompatibility groups, suggesting that other plasmid genes are spreading in K. pneumoniae isolates, which can coharbor and spread different carbapenemases simultaneously.

In Vitro Antimicrobial Activity of Five Newly Approved Antibiotics against Carbapenemase-Producing Enterobacteria-A Pilot Study in Bulgaria.

To solve the problem with pan-drug resistant and extensively drug-resistant Gram-negative microbes, newly approved drugs such as ceftazidime/avibactam, cefiderocol, plazomicin, meropenem/vaborbactam, and eravacycline have been introduced in practice. The aim of the present study was to collect carbapenemase-producing clinical Enterobacterales isolates, to characterize their carbapenemase genes and clonal relatedness, and to detect their susceptibility to commonly used antimicrobials and the above-mentioned newly approved antibiotics. Sixty-four carbapenemase producers were collected in a period of one year from four Bulgarian hospitals, mainly including Klebsiella pneumoniae (89% of the isolates) and also single Proteus mirabilis, Providencia stuartii and Citrobacter freundii isolates. The main genotype was blaNDM-1 (in 61%), followed by blaKPC-2 (23%), blaVIM-1 (7.8%) and blaOXA-48 (7.8%). Many isolates showed the presence of ESBL (blaCTX-M-15/-3 in 76.6%) and AmpC (blaCMY-4 in 37.5% or blaCMY-99 in 7.8% of isolates). The most common MLST type was K. pneumoniae ST11 (57.8%), followed by ST340 (12.5%), ST258 (6.3%) and ST101 (6.3%). The isolates were highly resistant to standard-group antibiotics, except they were susceptible to tigecycline (83.1%), colistin (79.7%), fosfomycin (32.8%), and aminoglycosides (20.3-35.9%). Among the newly approved compounds, plazomicin (90.6%) and eravacycline (76.3%) showed the best activity. Susceptibility to ceftazidime/avibactam and meropenem/vaborbactam was 34.4% and 27.6%, respectively. For cefiderocol, a large discrepancy was observed between the percentages of susceptible isolates according to EUCAST susceptibility breakpoints (37.5%) and those of CLSI (71.8%), detected by the disk diffusion method. This study is the first report to show patterns of susceptibility to five newly approved antibiotics among molecularly characterized isolates in Bulgaria. The data may contribute to both the improvement of treatment of individual patients and the choice of infection control strategy and antibiotic policy.

Molecular Characteristics and Antimicrobial Susceptibility Profiles of blaKPC-Producing Escherichia Coli Isolated from a Teaching Hospital in Shanghai, China.

Introduction: Carbapenem-Resistant Enterobacteriaceae (CRE) has posed a significant threat to humans.The aim of this study was to investigate the molecular characteristics of blaKPC-producing Escherichia coli in a university-affiliated tertiary hospital. Methods: Polymerase chain reaction (PCR) and BLAST+ software were used to detect the prevalence of blaKPC in E. coli and Klebsiella pneumoniae. Whole-genome sequencing was performed for the blaKPC-harboring clinical E. coli isolates. Antimicrobial resistance genes, MLSTs, KPC-carrying plasmid typing and genetic environment of blaKPC were analyzed. A maximum likelihood core single nucleotide polymorphism (SNP)-based phylogeny tree was constructed to determine the evolutionary relationships within this ST131 collection. Conjugation experiments were performed to determine the mobilization of blaKPC. The minimal inhibitory concentrations of the common antimicrobial agents were determined using the broth microdilution method. Results: The prevalence of blaKPC in 424 clinical E. coli isolates and 1636 E. coli strains from GenBank database were 2.2% (45/2060) whereas the detection rate of blaKPC in K. pneumoniae from the GenBank database was 29.8% (415/1394). The blaKPC-harboring conjugants exhibited resistance to multiple ß-lactams, except for cefepime-zidebactam and ceftazidime-avibactam. All blaKPC-carring E. coli isolates were susceptible to tigecycline and polymyxin B. ST131 was the dominant sequence type of blaKPC-carring E. coli, accounting for 40.0% (18/45). Most of the blaKPC-producing ST131 E. coli (89.5%,17/19) belonged to clade C ST131 lineage. Genetic environment analysis revealed that 57.8% (26/45) of blaKPC gene was linked to Tn4401-associated structure ISKpn6-blaKPC-ISKpn7. IncN was the most common plasmid type in KPC-producing E. coli whereas IncFII was the dominant plasmid type in KPC-producing K. pneumoniae. Conclusion: The detection rate of blaKPC was lower in E. coli compared with K. pneumoniae. The dominant sequence and plasmid types of blaKPC-harboring isolates differed between E. coli and K. pneumoniae. Further studies about the role of the defense system in acquisition of KPC-plasmids in E. coli will be performed to provide new insights into the low prevalence of blaKPC.

Treated municipal wastewater as a source of high-risk and emerging multidrug-resistant clones of E. coli and other Enterobacterales producing extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are a major public health problem, and wastewater from municipal wastewater treatment plants (WWTPs) is a potential means of spreading them into the environment and community. Our objective was to isolate ESBL-producing E. coli and other Enterobacterales from wastewater after treatment at Croatia's largest WWTP and to characterize these isolates by phenotypic and genotypic testing. Of the 200 bacterial isolates, 140 were confirmed as Enterobacterales by MALDI-TOF MS, with Escherichia coli and Klebsiella spp. predominating (69% and 7%, respectively). All 140 enterobacterial isolates were multidrug-resistant (MDR) and produced ESBLs. The most prevalent ESBL genes among the isolates tested were blaCTX-M-15 (60%), blaTEM-116 (44%), and blaCTX-M-3 (13%). Most isolates (94%) carried more than one ESBL gene in addition to blaCTX-M. Genes encoding plasmid-mediated AmpC, most notably blaEBC, were detected in 22% of isolates, whereas genes encoding carbapenemases (blaOXA-48, blaNDM-1, blaVIM-1) were less represented (10%). In E. coli, 9 different sequence types (ST) were found, with the emerging high-risk clones ST361 (serotype A-O9:H30) and pandemic ST131 (serotype B2-O25:H4) predominating (32% and 15%, respectively). Other high-risk E. coli clones included ST405 (3%), ST410 (3%), CC10 (3%), ST10 (3%), and ST38 (2%), and emerging clones included ST1193 (2%) and ST635 (2%). Whole-genome sequencing of three representative E. coli from two dominant clone groups (ST361 and ST131) and one extensively drug-resistant K. oxytoca revealed the presence of multiple plasmids and resistance genes to several other antibiotic classes, as well as association of the blaCTX-M-15 gene with transposons and insertion sequences. Our findings indicate that treated municipal wastewater contributes to the spread of emerging and pandemic MDR E. coli clones and other enterobacterial strains of clinical importance into the aquatic environment, with the risk of reintroduction into humans.

Rapid AST: Possibility of inferring resistance mechanisms with complex phenotypes.

The new automated systems designed for rapid performance of AST have significantly reduced the response time for susceptibility testing of microorganisms causing bacteremia and sepsis. The Accelerate Pheno® system (AAC) is one such system. Our objective for this study was to determine whether the AAC system is capable of providing an accurate susceptibility profile to infer resistance mechanisms in different carbapenemase-producing isolates when compared to the MicroScan WalkAway System (MWS). Disk diffusion method was also performed on all isolates as a reference method. Additionally, we compared the results obtained with the routine AST production system. We selected 19 isolates from the cryobank of the Microbiology department, all of which were carbapenemase-producing gram-negative bacilli. AAC was able to identify and infer the resistance of a total of 10 isolates, with an EA and CA of 84.2% for meropenem and 88.2% and 64.7% for ertapenem EA and CA, respectively. If we consider the disk diffusion technique, the CA was 57.9% and 76.5% for meropenem and ertapenem. However, in the presence of carbapenemases, AAC was not able to provide adequate MICs or infer the resistance mechanisms of the isolates accurately. Further studies with a larger number of isolates, including the new antibiotics ceftolozane/tazobactam and ceftazidime/avibactam, are needed for a more comprehensive comparison.