Cloacae Complex Research Articles

The EC-COMPASS: Long-term, multi-centre surveillance of Enterobacter cloacae complex – a clinical perspective

Enterobacter cloacae complex (ECCO) comprises closely related Enterobacterales, causing a variety of infections ranging from mild urinary tract infections to severe bloodstream infections. ECCO has emerged as a significant cause of healthcare-associated infections, particularly in neonatal and adult intensive care. The Enterobacter Cloacae COMplex PASsive Surveillance (EC-COMPASS) aims to provide a detailed multi-centre overview of ECCO epidemiology and resistance patterns detected in routine microbiological diagnostics in four German tertiary-care hospitals. In a sentinel cluster of four German tertiary-care hospitals, all culture-positive ECCO results between 1st January 2020 and 31st December 2022, were analysed based on Hybase® laboratory data. Analysis of 31,193 ECCO datasets from 14,311 patients revealed a higher incidence in male patients (P<0.05), although no significant differences were observed in ECCO infection phenotypes. The most common sources of ECCO were swabs (42.7%), urine (17.5%), respiratory secretions (16.1%), blood cultures (8.9%) and tissue samples (5.6%). The annual bacteraemia rate remained steady at approximately 33 cases per hospital. Invasive ECCO infections were predominantly found in oncology and intensive care units. Incidences of nosocomial outbreaks were infrequent and limited in scope. Notably, resistance to carbapenems was consistently low. EC-COMPASS offers a profound clinical perspective on ECCO infections in German tertiary-healthcare settings, highlighting elderly men in oncology and intensive care units as especially vulnerable to ECCO infections. Early detection strategies targeting at-risk patients could improve ECCO infection management.

A novel F type plasmid encoding mcr-10 in a clinical Enterobacter ludwigii strain from a tertiary hospital in the Czech Republic

ObjectiveHere we describe a novel IncFIA plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. MethodsThe strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using center for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST repsectively) and virulence genes using VFDB. ResultsΤhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and was susceptible to the 2nd and 3rd generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129,863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against NCBI database; no similar plasmid sequence was detected implying a novel plasmid structure. Nevertheless, it showed a partial similarity with: pRHBSTW-00123_3 and FDAARGOS 1432 which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the USA respectively, and pEC81-mcr which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for: curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins, and regulators of sigma factor. ConclusionIn conclusion, we identified a novel IncF plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis

A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Clinical and genomic characteristics of IMP-producing Enterobacter cloacae complex and Klebsiella pneumoniae.

Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-β-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae, and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae, respectively, and all isolates carried blaIMP-1. Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales. Among the infected patients, therapy varied and largely consisted of conventional β-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-β-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase (VIM).

Enterobacter asburiae ST229: an emerging carbapenemases producer

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. “Policlinico San Matteo” in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Evaluation of predictors of third-generation cephalosporin non-susceptibility and factors affecting recurrence or death in bacteremia caused by Citrobacter freundii complex, Enterobacter cloacae complex, and Klebsiella aerogenes

Factors involved in the susceptibility of third-generation cephalosporins (3GCs) to bacteremia caused by Citrobacter freundii complex, Enterobacter cloacae complex, and Klebsiella aerogenes were investigated based on a case-case-control design. Antimicrobial therapy administered 30 days prior to bacteremia and hospitalization within 90 days were common risk factors for the 3GC susceptible and 3GC non-susceptible groups, while hospitalization from an institution or another hospital was a specific risk factor for the 3GC non-susceptible group. We also attempted to examine the factors affecting the clinical outcome of bacteremia. Hospitalization more than 14 days before the onset of bacteremia was an independent factor indicating poor clinical outcome. In contrast, the implementation of source control was an independent predictor of successful treatment. Although a longer hospital stay before the onset of bacteremia was associated with worse clinical outcomes, implementation of source control may have contributed to improved treatment outcomes for bacteremia.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum β-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

The importance of meropenem resistance, rather than imipenem resistance, in defining carbapenem-resistant Enterobacterales for public health surveillance: an analysis of national population-based surveillance

BackgroundIn Japan, carbapenem-resistant Enterobacterales (CRE) infections were incorporated into the National Epidemiological Surveillance of Infectious Diseases (NESID) in 2014, necessitating mandatory reporting of all CRE infections cases. Subsequently, pathogen surveillance was initiated in 2017, which involved the collection and analysis of CRE isolates from reported cases to assess carbapenemase gene possession. In this surveillance, CRE is defined as (i) minimum inhibitory concentration (MIC) of meropenem ≥2 mg/L (MEPM criteria) or (ii) MIC of imipenem ≥2 mg/L and MIC of cefmetazole ≥64 mg/L (IPM criteria). This study examined whether the current definition of CRE surveillance captures cases with a clinical and public health burden.MethodsCRE isolates from reported cases were collected from the public health laboratories of local governments, which are responsible for pathogen surveillance. Antimicrobial susceptibility tests were conducted on these isolates to assess compliance with the NESID CRE definition. The NESID data between April 2017 and March 2018 were obtained and analyzed using antimicrobial susceptibility test results.ResultsIn total, 1681 CRE cases were identified during the study period, and pathogen surveillance data were available for 740 (44.0%) cases. Klebsiella aerogenes and Enterobacter cloacae complex were the dominant species, followed by Klebsiella pneumoniae and Escherichia coli. The rate of carbapenemase gene positivity was 26.5% (196/740), and 93.4% (183/196) of these isolates were of the IMP type. Meanwhile, 315 isolates were subjected to antimicrobial susceptibility testing. Among them, 169 (53.7%) fulfilled only the IPM criteria (IPM criteria-only group) which were susceptible to meropenem, while 146 (46.3%) fulfilled the MEPM criteria (MEPM criteria group). The IPM criteria-only group and MEPM criteria group significantly differed in terms of carbapenemase gene positivity (0% vs. 67.8%), multidrug resistance rates (1.2% vs. 65.8%), and mortality rates (1.8% vs 6.9%).ConclusionThe identification of CRE cases based solely on imipenem resistance has had a limited impact on clinical management. Emphasizing resistance to meropenem is crucial in defining CRE, which pose both clinical and public health burden. This emphasis will enable the efficient allocation of limited health and public health resources and preservation of newly developed antimicrobials.

Analysis of the transmission chain of carbapenem-resistant Enterobacter cloacae complex infections in clinical, intestinal and healthcare settings in Zhejiang province, China (2022–2023)

Considerable attention is given to intensive care unit-acquired infections; however, research on the transmission dynamics of multichain carbapenemase-resistant Enterobacter cloacae complex (CRECC) outbreaks remains elusive. A total of 118 non-duplicated CRECC strains were isolated from the clinical, intestinal, and hospital sewage samples collected from Zhejiang province of China during 2022–2023. A total of 64 CRECC strains were isolated from the hospital sewage samples, and their prevalence increased from 10.0 % (95 % confidence interval, CI = 0.52–45.8 %) in 2022 to 63.6 % (95 % CI = 31.6–87.6 %) in 2023. Species-specific identification revealed that Enterobacter hormaechei was the predominant CRECC species isolated in this study (53.4 %, 95 % CI = 44.0–62.6 %). The antimicrobial susceptibility profiles indicated that all 118 CRECC strains conferred high-level resistance to β-lactam antibiotics, ceftacillin/avibactam, and polymyxin. Furthermore, all CRECC strains exhibited resistance to β-lactams, quinolones, and fosfomycin, with a higher colistin resistance rate observed in the hospital sewage samples (67.2 %, 95 % CI = 54.2–78.1 %). Several antibiotic resistance genes were identified in CRECC strains, including Class A carbapenemases (blaKPC-2) and Class B carbapenemases (blaNDM-1/blaIMP), but not Class D carbapenemases. The WGS analysis showed that the majority of the CRECC strains carried carbapenemase-encoding genes, with blaNDM-1 being the most prevalent (86.9 %, 95 % CI = 77.4–92.9 %). Furthermore, sequence typing revealed that the isolated CRECC strains belonged to diverse sequence types (STs), among which ST418 was the most prevalent blaNDM-positive strain. The high risk of carbapenemase-producing ST418 E. hormaechei and the blaNDM-harboring IncFIB-type plasmid (81.4 %, 95 % CI = 72.9–87.7 %) were detected and emphasized in this study. This study provides valuable insights into the prevalence, antimicrobial resistance, genomic characteristics, and plasmid analysis of CRECC strains in diverse populations and environments. The clonal relatedness analysis showed sporadic clonal transmission of ST418 E. hormaechei strains, supporting inter-hospital transmission.

Azomycin Orchestrate Colistin-Resistant Enterobacter cloacae Complex's Colistin Resistance Reversal In Vitro and In Vivo.

The Enterobacter cloacae complex (ECC) is a group of nosocomial pathogens that pose a challenge in clinical treatment due to its intrinsic resistance and the ability to rapidly acquire resistance. Colistin was reconsidered as a last-resort antibiotic for combating multidrug-resistant ECC. However, the persistent emergence of colistin-resistant (COL-R) pathogens impedes its clinical efficacy, and novel treatment options are urgently needed. We propose that azomycin, in combination with colistin, restores the susceptibility of COL-R ECC to colistin in vivo and in vitro. Results from the checkerboard susceptibility, time-killing, and live/dead bacterial cell viability tests showed strong synergistic antibacterial activity in vitro. Animal infection models suggested that azomycin-colistin enhanced the survival rate of infected Galleria mellonella and reduced the bacterial load in the thighs of infected mice, highlighting its superior in vivo synergistic antibacterial activity. Crystal violet staining and scanning electron microscopy unveiled the in vitro synergistic antibiofilm effects of azomycin-colistin. The safety of azomycin and azomycin-colistin at experimental concentrations was confirmed through cytotoxicity tests and an erythrocyte hemolysis test. Azomycin-colistin stimulated the production of reactive oxygen species in COL-R ECC and inhibited the PhoPQ two-component system to combat bacterial growth. Thus, azomycin is feasible as a colistin adjuvant against COL-R ECC infection.

Advanced vaccinomic, immunoinformatic, and molecular modeling strategies for designing Multi- epitope vaccines against the Enterobacter cloacae complex.

The increasing and ongoing issue of antibiotic resistance in bacteria is of huge concern globally, mainly to healthcare facilities. It is now crucial to develop a vaccine for therapeutic and preventive purposes against the bacterial species causing hospital-based infections. Among the many antibiotic- resistant bacterial pathogens, the Enterobacter cloacae complex (ECC) including six species, E. Colcae, E. absuriae, E. kobie, E. hormaechei, E. ludwigii, and E. nimipressuralis, are dangerous to public health and may worsen the situation. Vaccination plays a vital role in the prevention of infections and infectious diseases. This research highlighted the construction and design of a multi-epitope vaccine for the E. cloacae complex by retrieving their complete sequenced proteome. The retrieved proteome was assessed to opt for potential vaccine candidates using immunoinformatic tools. Both B and T-cell epitopes were predicted in order to create both humoral and cellular immunity and further scrutinized for antigenicity, allergenicity, water solubility, and toxicity analysis. The final potential epitopes were subjected to population coverage analysis. Major histocompatibility complex (MHC) class combined, and MHC Class I and II world population coverage was obtained as 99.74%, and 98.55% respectively while a combined 81.81% was covered. A multi-epitope peptide-based vaccine construct consisting of the adjuvant, epitopes, and linkers was subjected to the ProtParam tool to calculate its physiochemical properties. The total amino acids were 236, the molecular weight was 27.64kd, and the vaccine construct was stable with an instability index of 27.01. The Grand Average of Hydropathy (GRAVY) (hydrophilicity) value obtained was -0.659, being more negative and depicting the hydrophilic character. It was non-allergen antigenic with an antigenicity of 0.8913. The vaccine construct was further validated for binding efficacy with immune cell receptors MHC-I, MHC-II, and Toll-like receptor (TLR)-4. The molecular docking results depict that the designed vaccine has good binding potency with immune receptors crucial for antigen presentation and processing. Among the Vaccine-MHC-I, Vaccine-MHC-II, and Vaccine-TLR-4 complexes, the best-docked poses were identified based on their lowest binding energy scores of -886.8, -995.6, and -883.6, respectively. Overall, we observed that the designed vaccine construct can evoke a proper immune response and the construct could help experimental researchers in the formulation of a vaccine against the targeted pathogens.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Hospital sewage in Brazil: a reservoir of multidrug-resistant carbapenemase-producing Enterobacteriaceae.

The One Health concept recognizes that human health is clearly linked to the health of animals and the environment. Infections caused by bacteria resistant to carbapenem antibiotics have become a major challenge in hospitals due to limited therapeutic options and consequent increase in mortality. In this study, we investigated the presence of carbapenem-resistant Enterobacteriaceae in 84 effluent samples (42 from hospital and 42 from non-hospital) from Campo Grande, midwest Brazil. First, sewage samples were inoculated in a selective culture medium. Bacteria with reduced susceptibility to meropenem and ertapenem were then identified and their antimicrobial susceptibility was determined using the Vitek-2 system. The blaKPC genes were detected using PCR and further confirmed by sequencing. Carbapenem-resistant Enterobacteriaceae (CRE) were identified in both hospital (n=32) and non-hospital effluent (n=16), with the most common being Klebsiella pneumoniae and of the Enterobacter cloacae complex species. This is the first study to indicate the presence of the blaKPC-2 gene in carbapenem-resistant Enterobacteriaceae, classified as a critical priority by the WHO, in hospital sewage in this region. The dissemination of carbapenem antibiotic-resistant genes may be associated with clinical pathogens. Under favorable conditions and microbial loads, resistant bacteria and antimicrobial-resistance genes found in hospital sewage can disseminate into the environment, causing health problems. Therefore, sewage treatment regulations should be implemented to minimize the transfer of antimicrobial resistance from hospitals.

Using metal nanoparticles (ZnO and Ag) to enhance DNA isolation and amplification

Recently, there have been significant improvements in the methods used to isolate and purify genomic DNA. The implementation of high-throughput facilities has accelerated the advancement of technology, enabling more rapid and effective DNA processing than ever before. The study's goal was to develop a cost-effective and efficient technique to enhance the characterization of isolated DNA that is suitable for subsequent molecular downstream applications. The DNA used as study object obtained from suspensions of pathogenic bacteria, Klebsiella pneumoniae (K. pneumoniae) and the Enterobacter cloacae complex (E. cloacae) using a manual salting out method. The method includes utilizing ZnO and Ag nanoparticles (Nps), to enhance isolation of genomic DNA. The study showed that using Nps at concentrations of 0.02 mg/ml for Ag and 0.004 mg/ml for ZnO led to better DNA quality and quantity compared to the control group without Nps, resulting in ideal outcomes. In addition, the presence of these Nps greatly increased the efficiency of PCR amplification for detecting the 16S ribosomal DNA (rDNA) of K. pneumoniae and E. cloacae. This resulted in the effective amplification of a 709 base pair segment in both type of Nps. The incorporation of nanoparticles technology into DNA extraction methods is in line with the current demands of the fast-progressing fields of molecular biology and biotechnology.

Genome sequence-based identification of Enterobacter strains and description of Enterobacter pasteurii sp. nov.

Accurate taxonomy is essential for microbial biological resource centers, since the microbial resources are often used to support new discoveries and subsequent research. Here, we used genome sequence data, alongside matrix-assisted laser desorption/ionization time-of-flight mass spectrometer biotyper-based protein profiling, to accurately identify six Enterobacter cloacae complex strains. This approach effectively identified distinct species within the E. cloacae complex, including Enterobacter asburiae, "Enterobacter xiangfangensis," and Enterobacter quasihormaechei. Moreover, the study revealed the existence of a novel species within the Enterobacter genus, for which we proposed the name Enterobacter pasteurii sp. nov. In summary, this study demonstrates the significance of adopting a genome sequence-driven taxonomy approach for the precise identification of bacterial strains in a biological resource center and expands our understanding of the E. cloacae complex.

300. Disturbance of Gut Microbial Diversity in ICU Patients from Colombia is Driven by Multi-Drug Resistant Organisms (MDRO) Colonization

Abstract Background Gut microbiota plays a critical role in health, with essential functions in physiology, metabolism, immunity, regulation, and protection against pathogens. Critically-ill patients admitted on the ICU experience perturbation in the microbiota, with reduction in the phylogenetic diversity and composition. Here, we aimed to investigate the rectal microbiota in patients admitted on the ICU from Colombia, focused on MDRO colonization. Methods Microbiome analysis and microbiological screening for MDRO and WGS were performed in rectal swabs taken on admission day from 180 patients from 5 ICUs. Microbiome analysis was determined by 16S rRNA sequencing using Illumina Miseq platform. alpha-diversity metrics were calculated by Richness, Simpson, and Shannon indexes, and beta-diversity was determined using Similarity Percentages (SIMPER) analysis. Patients were grouped into 2 cohorts: MDRO-colonized and non-colonized. Clinical data was collected using REDCap. Results MDRO colonization was detected in 13 patients (7%), and Enterobacter cloacae complex was the most prevalent microorganism (38%), followed by K. pneumoniae (23%), and P. aeruginosa (15%). Of these MDRO, 23% and 15% harbored KPC-2 and KPC-3/OXA-9 combination, respectively. Prevotella and Bacteroides were the most abundant genus in the microbiota of all patients. Interestingly, microbiota of MDRO-colonized patients showed lower diversity (Shannon p:0.0025, Simpson p: 0.0031 and richness 0.012) and had less bacterial taxa than the observed in the non-colonized group (n=167), in which Bacteroides, Porphyromonas, Campylobacter, and Oscillospiraceae genus were exclusively detected. Moreover, Parabacteroides was a genus exclusively identified in the MDRO colonized group. Further, 14 genus including Oscillospiraceae, Lactobacillus, and Faecalibacterium related to beneficial functions were lower in abundance from MDRO-colonized patients, whereas some pathobionts were present in high abundance in this group. Most of patients were men (58%); median age was 59 y/o. Charlson comorbidity index 4 (IQR=1-6). Conclusion Loss in microbial diversity, with low abundance of beneficial microorganisms were associated with MDRO gut colonization in ICU patients from Colombia. Disclosures Karen Melissa Ordoñez, n/a, Pfizer: Advisor/Consultant|Pfizer: Honoraria

2747. Aztreonam-Avibactam Susceptibility Testing Program for Metallo-β-Lactamase-Producing Enterobacterales collected through the Antimicrobial Resistance Laboratory Network, March 2019 to April 2023

Abstract Background Metallo-β-lactamase-producing Enterobacterales (MBL-E) are a major public health concern as they cause infections with few effective treatment options. Aztreonam-avibactam (AZA) is a β-lactam/β-lactamase inhibitor combination agent in the drug development pipeline that is active against MBL-E. Though clinical studies are ongoing, the effective components can be administered by combining two FDA-approved drugs: aztreonam and ceftazidime-avibactam. In 2019, CDC initiated a program in the Antimicrobial Resistance Laboratory Network (AR Lab Network), the Expanded Antimicrobial Susceptibility Testing Program for Hard-to-Treat Infections, to provide antimicrobial susceptibility testing (AST) of MBL-E for AZA. We provide a summary of the program’s results. Methods Seven AR Lab Network laboratories validated the use of a digital dispenser to create custom broth microdilution panels for AZA AST. Testing requests must be for clinical decision-making purposes. Isolates must be an Enterobacterales and have laboratory results positive for an MBL gene (i.e., blaNDM, blaVIM, or blaIMP), or be not susceptible to all β-lactams tested. AZA testing results for confirmed MBL-E were reported back to submitters within 3 working days from receipt of the isolate and shared with CDC. Results From March 2019 through April 2023, AZA AST was performed for 250 isolates: 103 Escherichia coli, 86 Klebsiella pneumoniae, 46 Enterobacter cloacae complex, 8 K. oxytoca, 2 Providencia rettgeri, 1 K. aerogenes, 1 Morganella morganii, 1 Proteus mirabilis, 1 E. hormaechei, and 1 Citrobacter amalonaticus. Carbapenemase genes detected were: 206 blaNDM, 24 blaNDM/blaOXA-48-like, 14 blaKPC/blaNDM, 2 blaIMP, 2 blaKPC/blaIMP, and 2 blaNDM/blaIMP. All isolates were resistant to ceftazidime-avibactam; 227/250 (90.8%) were not susceptible to aztreonam (≥8 µg/mL). For isolates not susceptible to aztreonam, the addition of avibactam resulted in a median 128-fold MIC reduction of aztreonam. The MIC range of AZA was ≤0.03/4 - &amp;gt;64/4 µg/mL. Overall, the MIC50 and MIC90 of AZA were 0.5/4 µg/mL and 8/4 µg/mL, respectively; the MIC50 and MIC90 for E. coli were higher than other species, 4/4 µg/mL and 16/4 µg/mL. Conclusion Our data suggest that AZA has potent in vitro activity against MBL-E collected in the United States. Disclosures All Authors: No reported disclosures

The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: a dockyard of β-lactamases periling nosocomial infections.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) constitutes a global public health threat challenging clinical treatment and infection control, especially in low- and middle-income countries such as India. We analyzed the antimicrobial susceptibility, major β-lactamase genes, plasmid profiles, and genetic relatedness to understand the molecular epidemiology of CRECC clinical isolates (n = 44) in West Bengal, India, during 2021-2022. The majority (> 55%) of the isolates were resistant to fluoroquinolones, aminoglycosides, and co-trimoxazole, even > 20% for tigecycline and > 35% were extensively drug-resistant. Co-β-lactamase production was categorized into twenty-seven types, importantly NDM (84%), OXA-48 (40%), TEM (61%), CTX-M (46%), OXA-1 (55%), and MIR (27%). The NDM-1 and OXA-181 were major variants with the first observations of NDM-24 and -29 variants in India. Wide-range of plasmids (2 to > 212kb) were harbored by the β-lactamase-producing isolates: small (91%), medium (27%), large (9%), and mega (71%). IncX3, ColE1, and HI2 were noted in about 30% of isolates, while IncF and R were carried by < 20% of isolates. The clonally diverse CRECC isolates were noted to cause cross-infections, especially at superficial site, bloodstream, and urinary-tract. This is the first molecular surveillance on CRECC in India. The study isolates serve as the dockyard of NDM, TEM, and CTX-M harboring a wide range of plasmids. The outcomes of the study may strengthen local and national policies for infection prevention and control practices, clarifying the genetic diversity among CRECC. Extensive genomic study may further intersect the relationships between these different plasmids, especially with their sizes, types, and antibiotic resistance markers.

Phenol Biodegradation by some Bacterial Species Isolated from Oil Contaminated Environments

Phenols are poisonous chemical molecules that affect human and other mammals as well as the aquatic environment, particularly higher species in fresh water environments. useing microorganisms through bioremediation is an efficient technique for reducing dangerous chemicals and detoxification of soil contaminated with hydrocarbons. The aim of this study is to investigate the efficiency of some local bacterial species to degrade phenol. Bacterial species were isolated from soils contaminated with hydrocarbons from Al-Diwaniyah and Al-Basrah governorates. The remaining concentration of phenol was examined usingthe 4-aminoantipyrine assay. The results showed that the rate of decomposition of Enterobacter cloacae complex was 100% after an incubation period of 120h, pantoea spp was 100% after an incubation period of 120h, Acinetobacter lwoffii was 100% after an incubation period of 144h, Aeromonas salmonicida was 100% after an incubation period 168h, Escherichia hermannii was 100% after an incubation period of 168h, Sphingomonas paucimobilis was 100% after an incubation period of 168h, Streptococcus thoraltensis was 100% after an incubation period of 168h, Bacillus licheniformis was 97.98%after an incubation period of 168h, Bacillus subtilis was 95.91% after an incubation period of 168h, and Bacillus firmus was 70.91% after an incubation period of 168h. The tested bacterial strains can offer a potential basis for bioremediation of phenol polluted locations.