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519300.9855Antibiotic resistance genes prediction via whole genome sequence analysis of Stenotrophomonas maltophilia. BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is the first dominant ubiquitous bacterial species identified from the genus Stenotrophomonas in 1943 from a human source. S. maltophilia clinical strains are resistance to several therapies, this study is designed to investigate the whole genome sequence and antimicrobial resistance genes prediction in Stenotrophomonas maltophilia (S. maltophilia) SARC-5 and SARC-6 strains, isolated from the nasopharyngeal samples of an immunocompromised patient. METHODS: These bacterial strains were obtained from Pakistan Institute of Medical Sciences (PIMS) Hospital, Pakistan. The bacterial genome was sequenced using a whole-genome shotgun via a commercial service that used an NGS (Next Generation Sequencing) technology called as Illumina Hiseq 2000 system for genomic sequencing. Moreover, detailed in-silico analyses were done to predict the presence of antibiotic resistance genes in S. maltophilia. RESULTS: Results showed that S. maltophilia is a rare gram negative, rod-shaped, non sporulating bacteria. The genome assembly results in 24 contigs (>500 bp) having a size of 4668,850 bp with 65.8% GC contents. Phylogenetic analysis showed that SARC-5 and SARC-6 were closely related to S. maltophilia B111, S. maltophilia BAB-5317, S. maltophilia AHL, S. maltophilia BAB-5307, S. maltophilia RD-AZPVI_04, S. maltophilia JFZ2, S. maltophilia RD_MAAMIB_06 and lastly with S. maltophilia sp ROi7. Moreover, the whole genome sequence analysis of both SARC-5 and SARC-6 revealed the presence of four resistance genes adeF, qacG, adeF, and smeR. CONCLUSION: Our study confirmed that S. maltophilia SARC-5 and SARC-6 are one of the leading causes of nosocomial infection which carry multiple antibiotic resistance genes.202438128408
140710.9850World Health Organization priority antimicrobial resistance in Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecium healthcare-associated bloodstream infections in Brazil (ASCENSION): a prospective, multicentre, observational study. BACKGROUND: Carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) are listed by World Health Organization (WHO) as priority antimicrobial-resistant bacteria. Data on WHO Priority Antimicrobial resistance Phenotype (WPAP) bacteria from low- and middle-income countries are scarce. In this study, we investigated the occurrence of WPAP in healthcare-associated bloodstream infections (BSI) in Brazil, an upper-middle-income country in South America. METHODS: ASCENSION was a prospective, multicentre, observational study conducted in 14 hospitals from four of five Brazilian regions. Enterobacterales, A. baumannii, P. aeruginosa, S. aureus and E. faecium BSIs in hospitalised patients were analysed. The primary outcome was the frequency of WPAP among all bacteria of interest. Secondary outcomes were incidence-density of bacteria isolates in hospitalised patients, WPAP proportions within bacterial species, and 28-day mortality. PCR for carbapenemase genes was performed in carbapenem-resistant Gram-negative bacteria. FINDINGS: Between August 15, 2022, and August 14, 2023, 1350 isolates (1220 BSI episodes) were included. WPAP accounted for 38.8% (n = 524; 95% Confidence Interval 32.0-46.1) of all isolates, with CRE (19.3%) as the most frequent, followed by CRAB (9.6%), MRSA (4.9%), VRE (2.7%), and CRPA (2.4%). Incidence-density of all and WPAP isolates were 1.91 and 0.77/1000 patients-day, respectively. Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the most common CRE, corresponding to 14.2% of all BSIs. A. baumannii isolates presented the highest proportion of WPAP (87.8%). Mortality rates were higher in patients with BSIs by WPAP than non-WPAP isolates. KPC (64.4%) was the predominant carbapenemase in CRE, followed by NDM (28.4%) and KPC + NDM co-production (7.1%). OXA-23 was the most frequent in CRAB. INTERPRETATION: A high frequency of WPAP bacteria, particularly CRKP and CRAB, were found in healthcare-associated BSIs in Brazil, posing them as a major public health problem in this country. FUNDING: National Council for Scientific and Technological Development, Brazil.202539957800
82820.9849Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana. Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and bla(CTX-M), bla(IMP), bla(GES), bla(VIM), bla(OXA-58)-like, bla(NDM), bla(OXA-23)-like, bla(OXA-48)-like and bla(KPC) occurring in descending order of frequency. The beta-lactamase genes bla(OXA-40/24)-like, bla(NMC_A/IMI), bla(BIC), bla(SME), bla(GIM) and bla(DIM) were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.202337623959
520630.9847Draft genome sequence of an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST644 isolated from a footpad infection in a Magellanic penguin (Spheniscus magellanicus). OBJECTIVES: The incidence of multidrug-resistant bacteria in wildlife animals has been investigated to improve our knowledge of the spread of clinically relevant antimicrobial resistance genes. The aim of this study was to report the first draft genome sequence of an extensively drug-resistant (XDR) Pseudomonas aeruginosa ST644 isolate recovered from a Magellanic penguin with a footpad infection (bumblefoot) undergoing rehabilitation process. METHODS: The genome was sequenced on an Illumina NextSeq(®) platform using 150-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.10, and the whole genome sequence was evaluated using bioinformatics approaches from the Center of Genomic Epidemiology, whereas an in-house method (mapping of raw whole genome sequence reads) was used to identify chromosomal point mutations. RESULTS: The genome size was calculated at 6436450bp, with 6357 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracyclines, quinolones and fosfomycin; in addition, mutations in the genes gyrA (Thr83Ile), parC (Ser87Leu), phoQ (Arg61His) and pmrB (Tyr345His), conferring resistance to quinolones and polymyxins, respectively, were confirmed. CONCLUSION: This draft genome sequence can provide useful information for comparative genomic analysis regarding the dissemination of clinically significant antibiotic resistance genes and XDR bacterial species at the human-animal interface.201829277728
520140.9847Complete genome of Enterobacter sichuanensis strain SGAir0282 isolated from air in Singapore. BACKGROUND: Enterobacter cloacae complex (ECC) bacteria, such as E. cloacae, E. sichuanensis, E. kobei, and E. roggenkampii, have been emerging as nosocomial pathogens. Many strains isolated from medical clinics were found to be resistant to antibiotics, and in the worst cases, acquired multidrug resistance. We present the whole genome sequence of SGAir0282, isolated from the outdoor air in Singapore, and its relevance to other ECC bacteria by in silico genomic analysis. RESULTS: Complete genome assembly of E. sichuanensis strain SGAir0282 was generated using PacBio RSII and Illumina MiSeq platforms, and the datasets were used for de novo assembly using Hierarchical Genome Assembly Process (HGAP) and error corrected with Pilon. The genome assembly consisted of a single contig of 4.71 Mb and with a G+C content of 55.5%. No plasmid was detected in the assembly. The genome contained 4371 coding genes, 83 tRNA and 25 rRNA genes, as predicted by NCBI's Prokaryotic Genome Annotation Pipeline (PGAP). Among the genes, the antibiotic resistance related genes were included: Streptothricin acetdyltransferase (SatA), fosfomycin resistance protein (FosA) and metal-dependent hydrolases of the beta-lactamase superfamily I (BLI). CONCLUSION: Based on whole genome alignment and phylogenetic analysis, the strain SGAir0282 was identified to be Enterobacter sichuanensis. The strain possesses gene clusters for virulence, disease and defence, that can also be found in other multidrug resistant ECC type strains.202032127921
247750.9846Evaluation of targeted next-generation sequencing for microbiological diagnosis of acute lower respiratory infection. PURPOSE: To evaluate the performance of targeted next-generation sequencing (tNGS) in pathogen detection in acute lower respiratory infection. METHODS: The retrospective study was conducted between July 2023 and May 2024 at the Yantai Yuhuangding Hospital. Patients with acute lower respiratory infections were included. Qualified sputum or bronchoalveolar lavage fluid samples were collected for tNGS and conventional microbiological tests(CMTs), including culture, staining, polymerase chain reaction (PCR), and reverse transcription-PCR (RT-PCR). The time required and cost were counted. RESULTS: A total of 968 patients were enrolled. Study analysis discovered 1,019 strains of bacteria, 259 strains of fungi, 302 strains of viruses, 76 strains of Mycoplasma pneumoniae, and two strains of Chlamydia psittaci using tNGS. In addition, tNGS also identified 39 mecA, four KPC, 19 NDM, and two OXA-48 genes. The positive rates for bacteria, fungi, viruses, mycoplasma, and chlamydia obtained using tNGS were significantly higher than those determined using traditional methods. Among them, tNGS showed high consistence with mycobacterium DNA test, influenza A (H1N1) virus nucleic acid test and COVID-19 nucleic acid test. Poor consistency between drug resistance genes and bacterial resistance phenotypes was found. In addition, tNGS also had advantages over traditional methods in terms of detection time and cost. CONCLUSION: Compared to traditional methods, tNGS had higher sensitivity in detecting bacteria, fungi, viruses, and other pathogens in acute lower respiratory infection, and also had the advantages of timeliness and cost-effectiveness, making it a promising method for guiding clinical diagnosis.202540901079
140260.9846Detection of β-lactam resistance genes in Gram-negative bacteria from positive blood cultures using a microchip-based molecular assay. BACKGROUND: Accurate detection of β-lactam resistance genes in bloodstream infections is critical for guiding antimicrobial therapy. This study evaluates the Alifax Gram-negative resistance (GNR) microchip assay for detecting β-lactam resistance genes directly from positive blood cultures (PBCs) for Gram-negative (GN) bacteria, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. METHODS: Simulated (n=146) and clinical (n=106) GN-PBC samples were tested for bla (KPC), bla (VIM), bla (NDM), bla (IMP), bla (OXA-23)-like, bla (OXA-48)-like, bla (SHV)-ESBL, bla (CTX-M-1/9) group, and bla (CMY-2)-like genes using the GNR microchip assay. Whole-genome sequencing (WGS) served as the reference assay for simulated samples and, selectively, for clinical samples. The bioMérieux BioFire Blood Culture Identification 2 (BCID2) panel assay was used as a comparator for clinical samples. RESULTS: The GNR microchip assay correctly identified 203 (99.5%) of 204 β-lactam resistance genes in simulated samples. One sample tested false negative for a bla (SHV)-ESBL gene but true positive for a bla (KPC) gene. In clinical samples, GNR results were concordant with BCID2 for 113 (100%) of 113 genes included in both assays. Additionally, the GNR assay detected bla (CMY-2) -like (n=6), bla (OXA-23)-like (n=5), and bla (SHV)-ESBL (n=2), which are not targeted by BCID2, all confirmed by WGS. In two β-lactam-resistant P. aeruginosa samples but negative by the GNR assay, WGS confirmed the absence of acquired β-lactam resistance genes, suggesting alternative resistance mechanisms. CONCLUSION: The GNR microchip assay demonstrated high concordance and broader β-lactam resistance gene coverage compared to BCID2, supporting its potential role in routine diagnostics. Further validation in larger, prospective studies is warranted.202540529307
147570.9845Evaluation of the FilmArray(®) Pneumonia Plus Panel for Rapid Diagnosis of Hospital-Acquired Pneumonia in Intensive Care Unit Patients. The FilmArray(®) Pneumonia plus Panel (FAPP) is a new multiplex molecular test for hospital-acquired pneumonia (HAP), which can rapidly detect 18 bacteria, 9 viruses, and 7 resistance genes. We aimed to compare the diagnosis performance of FAPP with conventional testing in 100 intensive care unit (ICU) patients who required mechanical ventilation, with clinically suspected HAP. A total of 237 samples [76 bronchoalveolar lavages (BAL(DS)) and 82 endotracheal aspirates (ETA(DS)) obtained at HAP diagnosis, and 79 ETA obtained during follow-up (ETA(TT))], were analyzed independently by routine microbiology testing and FAPP. 58 patients had paired BAL(DS) and ETA(DS). The positivity thresholds of semi-quantified bacteria were 10(3)-10(4) CFUs/mL or 10(4) copies/mL for BAL, and 10(5) CFUs/mL or copies/mL for ETA. Respiratory commensals (H. influenzae, S. aureus, E. coli, S. pneumoniae) were the most common pathogens. Discordant results for bacterial identification were observed in 33/76 (43.4%) BAL(DS) and 36/82 (43.9%) ETA(DS), and in most cases, FAPP identified one supplemental bacteria (23/33 BAL(DS) and 21/36 ETA(DS)). An absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. No linear relationship was observed between bin and CFUs/mL variables. Concordant results between paired BAL(DS) and ETA(DS) were obtained in 46/58 (79.3%) patients with FAPP. One of the 17 resistance genes detected with FAPP (mecA/C and MREJ) was not confirmed by conventional testing. Overall, FAPP enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. Implementing this strategy may allow clinicians to make more timely and informed decisions.202032983057
210580.9845Infections Caused by Antimicrobial Drug-Resistant Saprophytic Gram-Negative Bacteria in the Environment. BACKGROUND: Drug-resistance genes found in human bacterial pathogens are increasingly recognized in saprophytic Gram-negative bacteria (GNB) from environmental sources. The clinical implication of such environmental GNBs is unknown. OBJECTIVES: We conducted a systematic review to determine how often such saprophytic GNBs cause human infections. METHODS: We queried PubMed for articles published in English, Spanish, and French between January 2006 and July 2014 for 20 common environmental saprophytic GNB species, using search terms "infections," "human infections," "hospital infection." We analyzed 251 of 1,275 non-duplicate publications that satisfied our selection criteria. Saprophytes implicated in blood stream infection (BSI), urinary tract infection (UTI), skin and soft tissue infection (SSTI), post-surgical infection (PSI), osteomyelitis (Osteo), and pneumonia (PNA) were quantitatively assessed. RESULTS: Thirteen of the 20 queried GNB saprophytic species were implicated in 674 distinct infection episodes from 45 countries. The most common species included Enterobacter aerogenes, Pantoea agglomerans, and Pseudomonas putida. Of these infections, 443 (66%) had BSI, 48 (7%) had SSTI, 36 (5%) had UTI, 28 (4%) had PSI, 21 (3%) had PNA, 16 (3%) had Osteo, and 82 (12%) had other infections. Nearly all infections occurred in subjects with comorbidities. Resistant strains harbored extended-spectrum beta-lactamase (ESBL), carbapenemase, and metallo-β-lactamase genes recognized in human pathogens. CONCLUSION: These observations show that saprophytic GNB organisms that harbor recognized drug-resistance genes cause a wide spectrum of infections, especially as opportunistic pathogens. Such GNB saprophytes may become increasingly more common in healthcare settings, as has already been observed with other environmental GNBs such as Acinetobacter baumannii and Pseudomonas aeruginosa.201729164118
147790.9844Multicenter Evaluation of the BIOFIRE Blood Culture Identification 2 Panel for Detection of Bacteria, Yeasts, and Antimicrobial Resistance Genes in Positive Blood Culture Samples. Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S. Food & Drug Administration. Results obtained with the BIOFIRE BCID2 Panel were compared to standard-of-care (SoC) results, sequencing results, PCR results, and reference laboratory antimicrobial susceptibility testing results to evaluate the accuracy of its performance. Results for 1,093 retrospectively and prospectively collected positive blood culture samples were initially enrolled, and 1,074 samples met the study criteria and were included in the final analyses. The BIOFIRE BCID2 Panel demonstrated an overall sensitivity of 98.9% (1,712/1,731) and an overall specificity of 99.6% (33,592/33,711) for Gram-positive bacteria, Gram-negative bacteria and yeast targets which the panel is designed to detect. One hundred eighteen off-panel organisms, which the BIOFIRE BCID2 Panel is not designed to detect, were identified by SoC in 10.6% (114/1,074) of samples. The BIOFIRE BCID2 Panel also demonstrated an overall positive percent agreement (PPA) of 97.9% (325/332) and an overall negative percent agreement (NPA) of 99.9% (2,465/2,767) for antimicrobial resistance determinants which the panel is designed to detect. The presence or absence of resistance markers in Enterobacterales correlated closely with phenotypic susceptibility and resistance. We conclude that the BIOFIRE BCID2 Panel produced accurate results in this clinical trial.202337227281
1481100.9842Molecular versus conventional assay for diagnosis of hospital-acquired pneumonia in critically ill patients: a single center experience. PURPOSE: Lower respiratory tract infections are reported as one of top five causes of mortality and morbidity in the world. A bacterial etiology is often involved in HAP, most frequently from multidrug resistant gram-negative bacteria, and fast accurate diagnosis of etiologic agent(s) of LRTI is essential for an appropriate management. The aim of this retrospective study was to evaluate the analytical performance of Biofire Filmarray Pneumonia Plus for bacteria detection in bronchoalveolar lavage samples and the concordance of bacterial loads between BFPP and cultural gold standard methods. METHODS: A total of 111 BAL samples were obtained from 111 consecutive patients admitted to Intensive Care Unit of "Renato Dulbecco" Teaching Hospital of Catanzaro, from March 2023 to March 2024. RESULTS: Compared to conventional methods, BFPP showed a sensitivity of 99 % and a specificity of 64 %. The agreement between the two methods was assessed by calculating PPA and NPA, being 89 % and 95 %, respectively. The most common bacterial species identified at BFPP was Klebsiella pneumoniae, followed by Acinetobacter calcaceuticus-baumanii complex, Staphylococcus aureus and Pseudomonas aeruginosa. Bacterial load (CFU/ml) in relation to copy number detected by molecular analysis showed the best performance for value ≥10(6) copie/mL. About molecular mechanisms of resistance in comparison to phenotypic profiles, the highest level of performance was observed for presence of KPC genes, all isolates showing resistance to carbapenems, followed by OXA-48 like and NDM. CONCLUSION: The high concordance reported in this study between the identification of resistance genes and phenotypic indication can lead to an appropriate, fast and tailored antibiotic therapy.202540513663
3069110.9842The hospital sink drain biofilm resistome is independent of the corresponding microbiota, the environment and disinfection measures. In hospitals, the transmission of antibiotic-resistant bacteria (ARB) may occur via biofilms present in sink drains, which can lead to infections. Despite the potential role of sink drains in the transmission of ARB in nosocomial infections, routine surveillance of these drains is lacking in most hospitals. As a result, there is currently no comprehensive understanding of the transmission of ARB and the dissemination of antimicrobial resistance genes (ARGs) and associated mobile genetic elements (MGEs) via sink drains. This study employed a multifaceted approach to monitor the total aerobic bacteria as well as the presence of carbapenemase-producing Enterobacterales (CPEs), the microbiota and the resistome of sink drain biofilms (SDBs) and hospital wastewater (WW) of two separate intensive care units (ICUs) in the same healthcare facility in France. Samples of SDB and WW were collected on a monthly basis, from January to April 2023, in the neonatal (NICU) and the adult (AICU) ICUs of Grenoble Alpes University Hospital. In the NICU, sink drain disinfection with surfactants was performed routinely. In the AICU, routine disinfection is not carried out. Culturable aerobic bacteria were quantified on non-selective media, and CPEs were screened using two selective agars. Isolates were identified by MALDI-TOF MS, and antibiotic susceptibility testing (AST) was performed on Enterobacterales and P. aeruginosa. The resistome was analyzed by high-throughput qPCR targeting >80 ARGs and MGEs. The overall bacterial microbiota was assessed via full-length 16S rRNA sequencing. No CPEs were isolated from SDBs in either ICU by bacterial culture. Culture-independent approaches revealed an overall distinct microbiota composition of the SDBs in the two ICUs. The AICU SDBs were dominated by pathogens containing Gram-negative bacterial genera including Pseudomonas, Stenotrophomona, Klebsiella, and Gram-positive Staphylococcus, while the NICU SDBs were dominated by the Gram-negative genera Achromobacter, Serratia, and Acidovorax, as well as the Gram-positive genera Weisella and Lactiplantibacillus. In contrast, the resistome of the SDBs exhibited no significant differences between the two ICUs, indicating that the abundance of ARGs and MGEs is independent of microbiota composition and disinfection practices. The AICU WW exhibited more distinct aerobic bacteria than the NICU WW. In addition, the AICU WW yielded 15 CPEs, whereas the NICU WW yielded a single CPE. All the CPEs were characterized at the species level. The microbiota of the NICU and AICU WW samples differed from their respective SDBs and exhibited distinct variations over the four-month period:the AICU WW contained a greater number of genes conferring resistance to quinolones and integron integrase genes, whereas the NICU WW exhibited a higher abundance of streptogramin resistance genes. Our study demonstrated that the resistome of the hospital SDBs in the two ICUs of the investigated healthcare institute is independent of the microbiota, the environment, and the local disinfection measures. However, the prevalence of CPEs in the WW pipes collecting the waste from the investigated drains differed. These findings offer valuable insights into the resilience of resistance genes in SDBs in ICUs, underscoring the necessity for innovative strategies to combat antimicrobial resistance in clinical environments.202540483807
5200120.9841Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil. Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (bla (IND-13), bla (CIA-3), bla (TEM-116), bla (OXA-209), bla (VEB-15)), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.202235966843
836130.9840Cross-Sectional Assessment on Carbapenem-Resistant Gram-Negative Bacteria Isolated from Patients in Moldova. Information on the molecular epidemiology and carbapenem resistance mechanisms in Gram-negative bacterial isolates in Moldova is scarce. To close this knowledge gap, carbapenem-resistant Gram-negative bacteria were collected over an 11-month period in a routine diagnostic laboratory in Moldova. Antimicrobial susceptibility was phenotypically and genotypically assessed. Phylogenetic relationships were investigated and multi-locus sequence types were provided. The assessment indicated several clusters of phylogenetically closely related carbapenem-resistant Klebsiella pneumoniae (sequence types ST101, ST395 and ST377), Acinetobacter baumannii (ST2, ST19 and ST78) and Pseudomonas aeruginosa (ST357 and ST654) isolates next to a number of less frequently observed species and sequence types. A phylogenetic relationship to characterized isolates from neighboring Ukraine could be confirmed. Identified carbapenemase genes comprised bla(OXA-23), bla(OXA-72) and bla(GES-11) in A. baumannii, bla(KPC-3), bla(NDM-1) and bla(OXA-48) in K. pneumoniae, as well as bla(VIM-2) in Pseudomonas aeruginosa. In conclusion, the assessment suggested the spread of carbapenem-resistant Gram-negative bacteria in Moldova which were partly pre-described from neighboring Ukraine, as well as likely spill-over events, facilitating the regional spread of carbapenem-resistant clones. Several isolates with very high genomic similarity further support the hypothesis of likely regional transmission events driven by several evolutionary successful clonal lineages.202540005787
1478140.9840Multicenter Evaluation of the FilmArray Blood Culture Identification 2 Panel for Pathogen Detection in Bloodstream Infections. The FilmArray Blood Culture Identification 2 panel (BCID2; bioMérieux) is a fully automated PCR-based assay for identifying bacteria, fungi, and bacterial resistance markers in positive blood cultures (BC) in about 1 h. In this multicenter study, we evaluated the performance of the BCID2 panel for pathogen detection in positive BC. Conventional culture and BCID2 were performed in parallel at four tertiary-care hospitals. We included 152 positive BC-130 monomicrobial and 22 polymicrobial cultures-in this analysis. The BCID2 assay correctly identified 90% (88/98) of Gram-negative and 89% (70/79) of Gram-positive bacteria. Five bacterial isolates targeted by the BCID2 panel and recovered from five positive BC, including three polymicrobial cultures, were missed by the BCID2 assay. Fifteen isolates were off-panel organisms, accounting for 8% (15/182) of the isolates obtained from BC. The mean positive percent agreement between the BCID2 assay and standard culture was 97% (95% confidence interval, 95 to 99%), with agreement ranging from 67% for Candida albicans to 100% for 17 targets included in the BCID2 panel. BCID2 also identified the bla(CTX-M) gene in seven BC, including one for which no extended-spectrum β-lactamase (ESBL)-producing isolate was obtained in culture. However, it failed to detect ESBL-encoding genes in three BC. Two of the 18 mecA/C genes detected by the BCID2 were not confirmed. No carbapenemase, mecA/C, or MREJ targets were detected. The median turnaround time was significantly shorter for BCID2 than for culture. The BCID2 panel may facilitate faster pathogen identification in bloodstream infections. IMPORTANCE Rapid molecular diagnosis combining the identification of pathogens and the detection of antibiotic resistance genes from positive blood cultures (BC) can improve the outcome for patients with bloodstream infections. The FilmArray BCID2 panel, an updated version of the original BCID, can detect 11 Gram-positive bacteria, 15 Gram-negative bacteria, 7 fungal pathogens, and 10 antimicrobial resistance genes directly from a positive BC. Here, we evaluated the real-life microbiological performance of the BCID2 assay in comparison to the results of standard methods used in routine practice at four tertiary care hospitals.202336519852
1480150.9839Prospective observational pilot study of the T2Resistance panel in the T2Dx system for detection of resistance genes in bacterial bloodstream infections. Early initiation of antimicrobial therapy targeting resistant bacterial pathogens causing sepsis and bloodstream infections (BSIs) is critical for a successful outcome. The T2Resistance Panel (T2R) detects the following resistance genes within organisms that commonly cause BSIs directly from patient blood samples: bla(KPC), bla(CTXM-14/15), bla(NDM)/bla(/IMP)/bla(VIM), bla(AmpC), bla(OXA), vanA, vanB, and mecA/mecC. We conducted a prospective study in two major medical centers for the detection of circulating resistance genes by T2R in patients with BSIs. T2R reports were compared to antimicrobial susceptibility testing (AST), phenotypic identification, and standard molecular detection assays. Among 59 enrolled patients, 25 resistance genes were identified: bla(KPC) (n = 10), bla(NDM)/bla(/IMP)/bla(VIM) (n = 5), bla(CTXM-14/15) (n = 4), bla(AmpC) (n = 2), and mecA/mecC (n = 4). Median time-to-positive-T2R in both hospitals was 4.4 hours [interquartile range (IQR): 3.65-4.97 hours] in comparison to that for positive blood cultures with final reporting of AST of 58.34 h (IQR: 45.51-111.2 hours; P < 0.0001). The sensitivity of T2R to detect the following genes in comparison to AST was 100% for bla(CTXM-14/15), bla(NDM)/bla(/)(IMP)/bla(VIM), bla(AmpC), mecA/mecC and 87.5% for bla(KPC). When monitored for the impact of significant antimicrobial changes, there were 32 events of discontinuation of unnecessary antibiotics and 17 events of escalation of antibiotics, including initiation of ceftazidime/avibactam in six patients in response to positive T2R results for bla(KPC). In summary, T2R markers were highly sensitive for the detection of drug resistance genes in patients with bacterial BSIs, when compared with standard molecular resistance detection systems and phenotypic identification assays while significantly reducing by approximately 90% the time to detection of resistance compared to standard methodology and impacting clinical decisions for antimicrobial therapy. IMPORTANCE: This is the first reported study to our knowledge to identify key bacterial resistance genes directly from the bloodstream within 3 to 5 hours in patients with bloodstream infections and sepsis. The study further demonstrated a direct effect in modifying initial empirical antibacterial therapy in response to T2R signal to treat resistant bacteria causing bloodstream infections and sepsis.202438456690
1479160.9839BioFire FilmArray BCID2 versus VITEK-2 System in Determining Microbial Etiology and Antibiotic-Resistant Genes of Pathogens Recovered from Central Line-Associated Bloodstream Infections. Central line-associated bloodstream infection (CLABSI) is among the most serious hospital acquired infections. Therefore, the rapid detection of the causative microorganism is of crucial importance to allow for the appropriate antimicrobial therapy. In the present study, we analyzed the clinical performance of the BioFire FilmArray Blood Culture Identification 2 (BCID2) panel in the identification of 33 microbial species and 10 antibiotic resistance genes in comparison to the VITEK-2 system. A total of 104 blood specimens were included. The FilmArray BCID2 results were concordant with the VITEK-2 system in 69/97 specimens (71.1%). Non-concordance was either due to the detection of more pathogens by the FilmArray BCID2 23/28 (82%) or microbial species were misidentified 5/28 (18%). Hence, in comparison to the VITEK-2 system, the FilmArray BCID2 panel showed an overall sensitivity of 75.8% (95% CI, 66-83%) and an overall specificity of 98% (95% CI, 97-98.8%) in detecting microbial species. For the resistance genes, the FilmArray BCID was able to detect the presence of blaCTX-M gene in 23 Gram-negative isolates, blaNDM and blaOXA-48- like genes in 14 and 13 isolates, respectively. The mecA and mecC genes were found in 23 Staphylococcus species, while mecA, mecC and MREJ genes were found in 4 Staphylococcus aureus isolates. The sensitivity and specificity for detecting resistance genes by the FilmArray BCID2 was 90% (95% CI, 81.4-95%) and 99.6% (95% CI, 99-100%), respectively. As concluded, the present study emphasizes the high sensitivity and specificity of the FilmArray BCID2 in the rapid and reliable detection of different bacteria and fungi from positive blood culture bottles, as well as the accurate detection of various antibiotic resistance markers.202236358274
1437170.9838Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany. Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM), bla (OXA-40), bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51), bla (IMI), bla (FIM) and bla (DIM). We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM) and bla (OXA-40), while multiplex-2 included bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51) and bla (IMI).Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.202133448924
1538180.9838KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: bla(KPC-80), bla(KPC-81), bla(KPC-96) and bla(KPC-97). Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of bla(KPC-2) resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant bla(KPC) genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for bla(KPC-80), bla(KPC-96), and bla(KPC-97) by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including bla(TEM-1), bla(OXA-18) and bla(OXA-1), were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of bla(KPC-2) and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, bla(KPC-2). The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.202438319084
2216190.9838Ultrafast detection of β-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing. Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1 h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.202337850345