Performance comparison of BD Phoenix CPO detect panel with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Klebsiella pneumoniae isolates.

BACKGROUND: We aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L. RESULTS: According to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively. CONCLUSION: While the CPO panel's phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.

Infection with the multidrug-resistant Klebsiella pneumoniae New Delhi metallo-B-lactamase strain in patients with COVID-19: Nec Hercules contra plures?

Background: During the coronavirus disease 2019 (COVID-19) pandemic, in patients treated for SARS-CoV-2 infection, infections with the Klebsiella pneumoniae bacteria producing New Delhi metallo-B-lactamase (NDM) carbapenemase in the USA, Brazil, Mexico, and Italy were observed, especially in intensive care units (ICUs). This study aimed to assess the impact of Klebsiella pneumoniae NDM infection and other bacterial infections on mortality in patients treated in ICUs due to COVID-19. Methods: The 160 patients who qualified for the study were hospitalized in ICUs due to COVID-19. Three groups were distinguished: patients with COVID-19 infection only (N = 72), patients with COVID-19 infection and infection caused by Klebsiella pneumoniae NDM (N = 30), and patients with COVID-19 infection and infection of bacterial etiology other than Klebsiella pneumoniae NDM (N = 58). Mortality in the groups and chosen demographic data; biochemical parameters analyzed on days 1, 3, 5, and 7; comorbidities; and ICU scores were analyzed. Results: Bacterial infection, including with Klebsiella pneumoniae NDM type, did not elevate mortality rates. In the group of patients who survived the acute phase of COVID-19 the prolonged survival time was demonstrated: the median overall survival time was 13 days in the NDM bacterial infection group, 14 days in the other bacterial infection group, and 7 days in the COVID-19 only group. Comparing the COVID-19 with NDM infection and COVID-19 only groups, the adjusted model estimated a statistically significant hazard ratio of 0.28 (p = 0.002). Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups. Conclusion: In patients treated for SARS-CoV-2 infection acquiring a bacterial infection due to prolonged hospitalization associated with the treatment of COVID-19 did not elevate mortality rates. The data suggests that in severe COVID-19 patients who survived beyond the first week of hospitalization, bacterial infections, particularly Klebsiella pneumoniae NDM, do not significantly impact mortality. Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups.

Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to ß-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

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.

Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Inactivation of hydrogenase-3 leads to enhancement of 1,3-propanediol and 2,3-butanediol production by Klebsiella pneumoniae.

Klebsiella pneumoniae can use glucose or glycerol as carbon sources to produce 1,3-propanediol or 2,3-butanediol, respectively. In the metabolism of Klebsiella pneumoniae, hydrogenase-3 is responsible for H2 production from formic acid, but it is not directly related to the synthesis pathways for 1,3-propanediol and 2,3-butanediol. In the first part of this research, hycEFG, which encodes subunits of the enzyme hydrogenase-3, was knocked out, so K. pneumoniae ΔhycEFG lost the ability to produce H2 during cultivation using glycerol as a carbon source. As a consequence, the concentration of 1,3-propanediol increased and the substrate (glycerol) conversion ratio reached 0.587 mol/mol. Then, K. pneumoniae ΔldhAΔhycEFG was constructed to erase lactic acid synthesis which led to the further increase of 1,3-propanediol concentration. A substrate (glycerol) conversion ratio of 0.628 mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545 mol/mol). Furthermore, since adhE encodes an alcohol dehydrogenase that catalyzes ethanol production from acetaldehyde, K. pneumoniae ΔldhAΔadhEΔhycEFG was constructed to prevent ethanol production. Contrary to expectations, this did not lead to a further increase, but to a decrease in 1,3-propanediol production. In the second part of this research, glucose was used as the carbon source to produce 2,3-butanediol. Knocking out hycEFG had distinct positive effect on 2,3-butanediol production. Especially in K. pneumoniae ΔldhAΔadhEΔhycEFG, a substrate (glucose) conversion ratio of 0.730 mol/mol was reached, which is higher compared to wild type strain (0.504 mol/mol). This work suggests that the inactivation of hydrogenase-3 may have a global effect on the metabolic regulation of K. pneumoniae, leading to the improvement of the production of two industrially important bulk chemicals, 1,3-propanediol and 2,3-butanediol.

1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.

Characterization of KPC-2-producing Klebsiella pneumoniae and affected patients of a pediatric hospital in Brazil.

Carbapenemase-producing Klebsiella pneumoniae (CPKp) infections are important threats to pediatric populations. Thus, a retrospective study was conducted in a Brazilian reference pediatric hospital, and 26 CPKp isolates obtained from 23 patients were characterized. The affected population had important underlying diseases, reflecting previous hospitalization and antibiotic use. Most CPKp isolates were resistant to all antibiotic classes, and blaKPC-2 was the only carbapenemase-encoding gene. blaCTX-M-15 was common among the isolates, and modification or absence of the mgrB gene was the cause of polymyxin B resistance. Ten different sequence types were identified, and clonal complex 258 was prevalent. Alleles wzi50 and wzi64 were the most recurrent ones regarding K-locus type, with a remarkable contribution of the epidemic ST11/KL64 lineage as a colonizer. Our findings show that lineages associated with the pediatric population are similar to those found in adults, reinforcing the need for epidemiological surveillance to effectively implement prevention and control measures.

Efficacy of ceftazidime-avibactam in various combinations for the treatment of experimental osteomyelitis due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae.

BACKGROUND: Optimal treatment of carbapenemase-producing Enterobacterales (CPE) bone infections is poorly defined. This study evaluated the efficacy of the novel beta-lactam-beta-lactamase inhibitor-ceftazidime-avibactam (CAZ-AVI)-with different antibiotic combinations in an experimental model of CPE osteomyelitis. METHODS: KPC-99YC is a clinical strain of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae with intermediate susceptibility to meropenem (MIC 4 mg/L), gentamicin (MIC 0.25 mg/L), colistin (MIC 0.25 mg/L), fosfomycin (MIC 4 mg/L) and ceftazidime-avibactam (MIC 1 mg/L). Time-kill curves were performed at 4x MIC. Osteomyelitis was induced in rabbits by tibial injection of 2×108 CFU of KPC-99YC. Six groups started treatment 14 days later for 7 days: control, colistin, CAZ-AVI, CAZ-AVI plus gentamicin, CAZ-AVI plus colistin and CAZ-AVI plus fosfomycin. Antibiotic dosages were selected to simulate plasma concentrations obtained in humans. Treatment was evaluated according to bone cultures quantified in log10 CFU. RESULTS: In vitro, CAZ-AVI plus colistin or gentamicin were rapidly bactericidal in contrast with CAZ-AVI plus fosfomycin. In vivo, compared with controls, colistin alone (P = 0.045) and CAZ-AVI alone or in combination significantly lowered bone bacterial counts (P < 0.001). Bone sterilisation was achieved in 67% and 100% of animals with combinations of CAZ-AVI plus colistin or gentamicin (P = 0.001 and P < 0.001, respectively) whereas other treatments were no different from controls. CAZ-AVI plus gentamicin provided greater bone bacterial reduction than CAZ-AVI plus colistin (P = 0.033). No CAZ-AVI-resistant strains emerged in treated rabbits, regardless of combination. CONCLUSIONS: CAZ-AVI plus gentamicin was the best effective combination therapy. Combinations with CAZ-AVI appear to be a promising treatment of KPC-producing Klebsiella pneumoniae osteomyelitis.

Antibiotic resistance patterns of carbapenemase-producing Enterobacterales in Mohammed VI University Hospital of Marrakech, Morocco.

OBJECTIVES: The emergence and spread of Carbapenem-Resistant Enterobacterales (CRE) has become a growing concern for health services, internationally, nationally, and regionally. In Morocco, the situation is more worrisome as studies on CRE are scarce and/or scattered and/or outdated. As a result, we carried out the present study to determine and update CRE prevalence at Mohammed VI University Hospital of Marrakech, Morocco. PATIENTS AND METHODS: A cross-sectional prospective study was carried out from March 2018 to March 2020 on 41161 clinical specimens of 23,469 patients suspected of bacterial infections. Enterobacterales strains were isolated following standard bacteriological procedures. Bacterial strains were identified using BD-Phoenix and MALDI-TOF-MS. Antibiotic susceptibility was determined for 14 antibiotics. Carbapenemase production and phenotypic detection were characterized using modified carbapenem inactivation phenotypic and immunochromatographic methods. RESULTS: All in all, 484 Enterobaterales resistant to at least one carbapenem were recovered. The majority was isolated from the neonatal unit (14%), followed by the urology-nephrology (11%), and plastic surgery departments (10%). K. pneumoniae (n=232) was the most isolated, followed by E. cloacae (n=148), E. coli (n=56), and S. marcescens (n=17). Antibiotic susceptibility profile showed high rates of resistance to ciprofloxacin (75.21%), gentamicin (84.50%), and cotrimoxazole (88.42%). Out of 484 CRE positive cultures, 388 (80.16%) were Carbapenemase-positive. Out of the latter, 170 were metallo-beta-lactamase producers (NDM), 162 OXA-48-like, and 56 both. CONCLUSION: These findings emphasize the urgent need for control precautions and strict measures to contain and mitigate this issue.

Structural characterization of the urease accessory protein UreF from Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that mostly affects those with weakened immune systems. Urease is a vital enzyme that can hydrolyze urea to ammonia and carbon dioxide as a source of nitrogen for growth. Urease is also a K. pneumoniae virulence factor that enables survival of the bacterium under nutrient-limiting conditions. UreF, an important nickel-binding urease accessory protein, is involved in the insertion of Ni2+ into the active site of urease. Here, the crystal structure of UreF from K. pneumoniae (KpUreF) is reported. Functional data show that KpUreF forms a stable dimer in solution. These results may provide a starting point for the design of urease inhibitors.

Methodology Establishment and Application of VITEK Mass Spectrometry to Detect Carbapenemase-Producing Klebsiella pneumoniae.

The ability of VITEK mass spectrometry (MS) in detection of bacterial resistance is currently under exploration and evaluation. In this study, we developed and validated a VITEK MS method to rapidly test carbapenemase-producing Klebsiella pneumoniae (CPKP). Solvents, antibiotic concentrations, crystal conditions and times, centrifugation speeds, and other factors were optimized to design a rapid sample pretreatment process for CPKP detection by VITEK MS. The related parameters of the mass spectrum were adjusted on the instrument to establish an CPKP detection mode. 133 clinically isolated strains of CPKP in the microbiology laboratory at the Shenzhen People's Hospital from 2004 to 2017 were selected for accuracy evaluation. The fresh suspected strains from the microbiology laboratory in 2020 were used to complete the clinical verification. Two antibiotics, meropenem (MEM) and imipenem (IPM), were used as substrates. These two substrates were incubated with suspected CPKP, and the results were obtained by VITEK MS detection. Using this method, different types of CPKP showed different detection results and all the CPKP strains producing KPC-2 and IMP-4 carbapenemase were detected by VITEK MS. Thus, VITEK MS can be used for rapid detection of CPKP, especially for some common types of CPKP. This method provides high accuracy and speed of detection. Combined with its cost advantages, it can be intensely valuable in clinical microbiology laboratories after the standard operating procedures are determined.

Drug Repurposing of the Unithiol: Inhibition of Metallo-ß-Lactamases for the Treatment of Carbapenem-Resistant Gram-Negative Bacterial Infections.

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.

Diffusion of OXA-48 carbapenemase among urinary isolates of Klebsiella pneumoniae in non-hospitalized elderly patients.

BACKGROUND: Recently, a dramatic increase of Klebsiella pneumoniae positive for OXA-48 ß-lactamases was observed first in the hospital setting and later in the long-term care facilities (LTCFs) and community in the Zagreb County, particularly, in urinary isolates. The aim of the study was to analyse the epidemiology and the mechanisms of antibiotic resistance of OXA-48 carbapenemase producing K. pneumoniae strains isolated from urine of non-hospitalized elderly patients. RESULTS: The isolates were classified into two groups: one originated from the LTCFs and the other from the community. Extended-spectrum ß-lactamases (ESBLs) were detected by double disk-synergy (DDST) and combined disk tests in 55% of the isolates (51/92). The ESBL-positive isolates exhibited resistance to expanded-spectrum cephalosporins (ESC) and in majority of cases to gentamicin. LTCFs isolates showed a significantly lower rate of additional ESBLs and consequential resistance to ESC and a lower gentamicin resistance rate compared to the community isolates, similarly to hospital isolates in Zagreb, pointing out to the possible transmission from hospitals.ESBL production was associated with group 1 of CTX-M or SHV-12 ß-lactamases. Ertapenem resistance was transferable from only 12 isolates. blaOXA-48 genes were carried by IncL plasmid in 42 isolates. In addition IncFII and IncFIB were identified in 18 and 2 isolates, respectively. Two new sequence types were reported: ST4870 and ST4781. CONCLUSIONS: This study showed eruptive and extensive diffusion of OXA-48 carbapenemase to LTCFs and community population in Zagreb County, particularly affecting patients with UTIs and urinary catheters. On the basis of susceptibility testing, ß-lactamase production, conjugation experiments, MLST and plasmid characterization it can be concluded that there was horizontal gene transfer between unrelated isolates, responsible for epidemic spread of OXA-48 carbapenemase in the LTCFs and the community The rapid spread of OXA-48 producing K. pneumoniae points out to the shortcomings in the infection control measures.

Prior Carriage Predicts Intensive Care Unit Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae.

Intensive care unit-acquired infection (ICU-AI) and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) carriage are a major concern worldwide. Our objective was to investigate the impact of ESBL-PE carriage on ICU-AI. Our study was prospective, observational, and noninterventional. It was conducted over a 5-year period (Jan 2013-Dec 2017) in the medical-surgical intensive care unit of the Cayenne General Hospital (French Amazonia). During the study period, 1,340 patients were included, 271 (20.2%) developed ICU-AI, and 16.2% of these were caused by ESBL-PE. The main sites of ICU-AI were ventilator-associated pneumonia (35.8%) and primary bloodstream infection (29.8%). The main responsible microorganisms were Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae (ESBL-P in 35.8% of isolates), and Enterobacter cloacae (ESBL-P in 29.8% of isolates). Prior ESBL-PE carriage was diagnosed in 27.6% of patients with ICU-AI. In multivariable analysis, the sole factor associated with ESBL-PE as the responsible organism of ICU-AI was ESBL-PE carriage before ICU-AI (P < 0.001; odds ratio: 7.9 95% CI: 3.4-18.9). ESBL-PE carriers (74 patients) developed ICU-AI which was caused by ESBL-PE in 32 cases (43.2%). This proportion of patients carrying ESBL-PE who developed ICU-AI to the same microorganism was 51.2% in ESBL-P K. pneumoniae, 5.6% in ESBL-P Escherichia coli, and 40% in ESBL-P Enterobacter spp. NPV of ESBL-PE carriage to predict ICU-AI caused by ESBL-PE was above 94% and PPV was above 43%. Carriage of ESBL-P K pneumoniae and Enterobacter spp. is a strong predictor of ICU-AI caused by these two microorganisms.