Pipeline validation for the identification of antimicrobial-resistant genes in carbapenem-resistant Klebsiella pneumoniae.

Antimicrobial-resistant Klebsiella pneumoniae is a global threat to healthcare and an important cause of nosocomial infections. Antimicrobial resistance causes prolonged treatment periods, high mortality rates, and economic impacts. Whole Genome Sequencing (WGS) has been used in laboratory diagnosis, but there is limited evidence about pipeline validation to parse generated data. Thus, the present study aimed to validate a bioinformatics pipeline for the identification of antimicrobial resistance genes from carbapenem-resistant K. pneumoniae WGS. Sequences were obtained from a publicly available database, trimmed, de novo assembled, mapped to the K. pneumoniae reference genome, and annotated. Contigs were submitted to different tools for bacterial (Kraken2 and SpeciesFinder) and antimicrobial resistance gene identification (ResFinder and ABRicate). We analyzed 201 K. pneumoniae genomes. In the bacterial identification by Kraken2, all samples were correctly identified, and in SpeciesFinder, 92.54% were correctly identified as K. pneumoniae, 6.96% erroneously as Pseudomonas aeruginosa, and 0.5% erroneously as Citrobacter freundii. ResFinder found a greater number of antimicrobial resistance genes than ABRicate; however, many were identified more than once in the same sample. All tools presented 100% repeatability and reproducibility and > 75% performance in other metrics. Kraken2 was more assertive in recognizing bacterial species, and SpeciesFinder may need improvements.

Association Between Gut Microbiota and Delirium in Acutely Ill Older Adults.

Our aim was to investigate the association between gut microbiota and delirium occurrence in acutely ill older adults. We included 133 participants 65+ years consecutively admitted to the emergency department of a tertiary university hospital, between September 2019 and March 2020. We excluded candidates with ≥24-hour antibiotic utilization on admission, recent prebiotic or probiotic utilization, artificial nutrition, acute gastrointestinal disorders, severe traumatic brain injury, recent hospitalization, institutionalization, expected discharge ≤48 hours, or admission for end-of-life care. A trained research team followed a standardized interview protocol to collect sociodemographic, clinical, and laboratory data on admission and throughout the hospital stay. Our exposure measures were gut microbiota alpha and beta diversities, taxa relative abundance, and core microbiome. Our primary outcome was delirium, assessed twice daily using the Confusion Assessment Method. Delirium was detected in 38 participants (29%). We analyzed 257 swab samples. After adjusting for potential confounders, we observed that a greater alpha diversity (higher abundance and richness of microorganisms) was associated with a lower risk of delirium, as measured by the Shannon (odds ratio [OR] = 0.77; 95% confidence interval [CI] = 0.60-0.99; p = .042) and Pielou indexes (OR = 0.69; 95% CI = 0.51-0.87; p = .005). Bacterial taxa associated with pro-inflammatory pathways (Enterobacteriaceae) and modulation of relevant neurotransmitters (Serratia: dopamine; Bacteroides, Parabacteroides: GABA) were more common in participants with delirium. Gut microbiota diversity and composition were significantly different in acutely ill hospitalized older adults who experienced delirium. Our work is an original proof-of-concept investigation that lays a foundation for future biomarker studies and potential therapeutic targets for delirium prevention and treatment.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. OBJECTIVE: To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. RESULTS: All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8-16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. CONCLUSION: VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Phenotypic and genotypic characteristics of a carbapenem-resistant Serratia marcescens cohort and outbreak: describing an opportunistic pathogen.

Serratia marcescens is an emerging opportunistic pathogen with high genetic diversity. This article describes the microbiological characteristics of isolates and the risk factors for infections caused by carbapenem-resistant S. marcescens. A retrospective study of patients colonized (n=43) and infected (n=20) with carbapenem-resistant S. marcescens over a 3-year period was conducted. Polymerase chain reaction for carbapenemase genes and molecular typing of all available strains was performed. Forty-two isolates were analysed, including three environmental samples identified during an outbreak. Thirty-five carbapenem-resistant S. marcescens carried blaKPC-2, one isolate was blaNDM-positive and four isolates carried blaOXA-101. The genomes were grouped into three clusters with 100% bootstrap; three patterns of mutations on ompC and ompF were found. The strains carried virulence genes related to invasion and haemolysis, and the environmental strains presented fewer mutations on the virulence genes than the clinical strains. Multi-variate analysis showed that previous use of polymyxin (P=0.008) was an independent risk factor for carbapenem-resistant S. marcescens infection. This study highlighted that blaKPC-2 in association with ompC or ompF mutation was the most common mechanism of resistance in the study hospital, and that previous use of polymyxin was an independent risk factor for carbapenem-resistant S. marcescens. There was a predominant clone, including the environmental isolates, suggesting that cross-transmission was involved in the dissemination of this pathogen.

Clinical outcome from hematopoietic cell transplant patients with bloodstream infection caused by carbapenem-resistant P. aeruginosa and the impact of antimicrobial combination in vitro.

Bloodstream infection (BSI) caused by carbapenem-resistant P. aeruginosa (CRPA) has high mortality in hematopoietic stem cell transplant (HSCT) recipients. We performed MIC, checkerboard, time-kill assay, PFGE, PCR, and whole genome sequence and described the clinical outcome through Epi Info comparing the antimicrobial combination in vitro. Mortality was higher in BSI caused by CRPA carrying the lasB virulence gene. The isolates were 97% resistant to meropenem displaying synergistic effect to 57% in combination with colistin. Seventy-three percent of the isolates harbored blaSPM-1 and Tn4371 and belonged to ST277. The synergistic effect in vitro with meropenem with colistin appeared to be a better therapeutic option.

Colistin-resistant Escherichia coli belonging to different sequence types: genetic characterization of isolates responsible for colonization, community- and healthcare-acquired infections.

The plasmid-mediated colistin-resistance gene named mcr-1 has been recently described in different countries and it became a public health challenge. Of note, few studies have addressed the spread of Escherichia coli harboring the mcr-1 gene in both, community and hospital settings. A total of seven colistin-resistant E. coli carrying mcr-1, collected from 2016 to 2018, from community (n=4), healthcare-acquired infections (n=2) and colonization (n=1) were identified in three high complexity hospitals in Sao Paulo, Brazil. These colistin-resistant isolates were screened for mcr genes by PCR and all strains were submitted to Whole Genome Sequencing and the conjugation experiment. The seven strains belonged to seven distinct sequence types (ST744, ST131, ST69, ST48, ST354, ST57, ST10), and they differ regarding the resistance profiles. Transference of mcr-1 by conjugation to E. coli strain C600 was possible in five of the seven isolates. The mcr-1 gene was found in plasmid types IncX4 or IncI2. Three of the isolates have ESBL-encoding genes (blaCTX-M-2, n=2; blaCTX-M-8, n=1). We hereby report genetically distinct E. coli isolates, belonging to seven STs, harboring the mcr-1 gene, associated to community and healthcare-acquired infections, and colonization in patients from three hospitals in Sao Paulo. These findings point out for the potential spread of plasmid-mediated colistin-resistance mechanism in E. coli strains in Brazil.

Gut Dysbiosis in Chagas Disease. A Possible Link to the Pathogenesis.

Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi. Cardiomyopathy and damage to gastrointestinal tissue are the main disease manifestations. There are data suggesting that the immune response to T. cruzi depends on the intestinal microbiota. We hypothesized that Chagas disease is associated with an altered gut microbiome and that these changes are related to the disease phenotype. The stool microbiome from 104 individuals, 73 with Chagas disease (30 with the cardiac, 11 with the digestive, and 32 with the indeterminate form), and 31 healthy controls was characterized using 16S rRNA amplification and sequencing. The QIIME (Quantitative Insights Into Microbial Ecology) platform was used to analyze the data. Alpha and beta diversity indexes did not indicate differences between the groups. However, the relative abundance of Verrucomicrobia, represented primarily by the genus Akkermansia, was significantly lower in the Chagas disease groups, especially the cardiac group, compared to the controls. Furthermore, differences in the relative abundances of Alistipes, Bilophila, and Dialister were observed between the groups. We conclude that T. cruzi infection results in changes in the gut microbiome that may play a role in the myocardial and intestinal inflammation seen in Chagas disease.

Alternative drugs against multiresistant Gram-negative bacteria.

OBJECTIVES: Enterobacterales and other non-fermenting Gram-negative bacteria have become a threat worldwide owing to the frequency of multidrug resistance in these pathogens. On the other hand, efficacious therapeutic options are quickly diminishing. The aims of this study were to describe the susceptibility of 50 multiresistant Gram-negative bacteria, mostly pan-resistant, against old and less-used antimicrobial drugs and to investigate the presence of antimicrobial resistance genes. METHODS: A total of 50 genetically distinct isolates were included in this study, including 14 Acinetobacter baumannii (belonging to ST79, ST317, ST835 and ST836), 1 Pseudomonas aeruginosa (ST245), 8 Serratia marcescens and 27 Klebsiella pneumoniae (belonging to ST11, ST340, ST258, ST16, ST23, ST25, ST101, ST234, ST437 and ST442). The isolates were submitted to antimicrobial susceptibility testing and whole-genome sequencing to evaluate lineages and resistance genes. RESULTS: Our results showed that some strains harboured carbapenemase genes, e.g. blaKPC-2 (28/50; 56%) and blaOXA-23 (11/50; 22%), and other resistance genes encoding aminoglycoside-modifying enzymes (49/50; 98%). Susceptibility rates to tigecycline (96%) in all species (except P. aeruginosa), to minocycline (100%) and doxycycline (93%) in A. baumannii, to ceftazidime/avibactam in S. marcescens (100%) and K. pneumoniae (96%), and to fosfomycin in S. marcescens (88%) were high. Chloramphenicol and quinolones (6% susceptibility each) did not perform well, making their use in an empirical scenario unlikely. CONCLUSIONS: This study involving genetically distinct bacteria showed promising results for tigecycline for all Gram-negative bacteria (except P. aeruginosa), and there was good activity of minocycline against A. baumannii, ceftazidime/avibactam against Enterobacterales, and fosfomycin against S. marcescens.

Comparison of methods for the detection of in vitro synergy in multidrug-resistant gram-negative bacteria.

BACKGROUND: The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. RESULTS: Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. CONCLUSIONS: The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories.

Multidrug-resistant Klebsiella pneumoniae: genetic diversity, mechanisms of resistance to polymyxins and clinical outcomes in a tertiary teaching hospital in Brazil.

Increased resistance to polymyxin in Klebsiella pneumoniae (ColRKP) has been observed. Molecular epidemiology, as well as the clinical impact of these difficult to treat pathogens need to be better characterized. We present the clinical outcomes of 28 patients infected by ColRKP in a tertiary hospital. Isolates with MIC >2 by Vitek 2 were confirmed by the microdilution broth test. Polymerase chain reaction (PCR) was performed for blaKPC, blaNDM, blaOXA-48 and blamcr-1 genes in the isolates, and Whole Genome Sequencing (WGS) was performed in six isolates. Seventeen (61%) patients were female and the mean age was 50 years old. In-hospital and 30-day mortality were 64% (18/28) and 53% (15/28), respectively. Central line-associated bloodstream infection in addition to bacteremia episodes due to other sources were the most frequent (61%). Mean APACHE and Charlson comorbidity index were 16 and 5, respectively. Twenty patients (71%) received at least one active drug and ten (35%) received two drugs: tigecycline 46% (13/28); amikacin 21% (6/28) and fosfomycin 3% (1 case). Twenty-six out of 28 tested cases were positive for blaKPC. Eight different clusters were identified. Four STs were detected (ST11, ST23, ST340, and ST437). Mutations on pmrA, arnB, udg, and yciM genes were present in all six isolates submitted to WGS; lpxMand mgrB mutations were also detected in all but one isolate. In conclusion, we observed resistance to polymyxin in severely ill patients mostly from intensive care units and/or immunosuppressed patients with high mortality rates in whom a diversity of ColRKP clusters was identified and might indicate selective pressure.

Bordetella trematum infection: case report and review of previous cases.

BACKGROUND: Bordetella trematum is an infrequent Gram-negative coccobacillus, with a reservoir, pathogenesis, a life cycle and a virulence level which has been poorly elucidated and understood. Related information is scarce due to the low frequency of isolates, so it is important to add data to the literature about this microorganism. CASE PRESENTATION: We report a case of a 74-year-old female, who was referred to the hospital, presenting with ulcer and necrosis in both legs. Therapy with piperacillin-tazobactam was started and peripheral artery revascularization was performed. During the surgery, a tissue fragment was collected, where Bordetella trematum, Stenotrophomonas maltophilia, and Enterococcus faecalis were isolated. After surgery, the intubated patient was transferred to the intensive care unit (ICU), using vasoactive drugs through a central venous catheter. Piperacillin-tazobactam was replaced by meropenem, with vancomycin prescribed for 14 days. Four days later, levofloxacin was added for 24 days, aiming at the isolation of S. maltophilia from the ulcer tissue. The necrotic ulcers evolved without further complications, and the patient's clinical condition improved, leading to temporary withdrawal of vasoactive drugs and extubation. Ultimately, however, the patient's general condition worsened, and she died 58 days after hospital admission. CONCLUSIONS: Despite being a rare finding, B. trematum is typically associated with the clinical manifestation of disorders that predispose to ulcer development, which can be infected by microorganisms. The combination of antibiotic therapy and surgical debridement plays a key role in preventing systemic infections. Monitoring the appearance of new cases of B. trematum is essential, since it can be an emerging microorganism. Isolating and defining the clinical relevance of unusual bacteria yields a more accurate perspective in the development of new diagnostic tools and allows for assessment of proper antimicrobial therapy.

Simultaneous colonization by Escherichia coli and Klebsiella pneumoniae harboring mcr-1 in Brazil.

CASE PRESENTATION: We present a case report of a woman, concurrently colonized by polymyxin-resistant E. coli and K. pneumoniae. A Brazilian female patient, in her mid-fifties, was hospitalized with schistosomiasis. During hospitalization, polymyxin-resistant E. coli and K. pneumoniae were isolated from surveillance cultures. METHODS: Identification, antimicrobial susceptibility testings, PCR for mcr-1, plasmid transfer by conjugation and whole genome sequencing were performed. RESULTS: E. coli ST744 and K. pneumoniae ST101 carrying mcr-1 gene were described. Transconjugant E. coli was positive for mcr-1 and IncX4 by PCR. The plasmid is a 33,304-base pair plasmid, and the mcr-1 gene was the only antimicrobial resistance gene present in the plasmid. CONCLUSIONS: This study presents a case report of a hospitalized woman, concurrently colonized by mcr-1-harboring E. coli ST744, a different ST from previously described in Brazil, and a K. pneumoniae ST101.

Polymyxin-resistant Pseudomonas aeruginosa assigned as ST245: First report in an intensive care unit in São Paulo, Brazil.

OBJECTIVES: Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe infections, especially in hospitalised and immunocompromised patients. Polymyxins are the last therapeutic option to treat infections caused by this micro-organism. Here we describe a polymyxin-resistant P. aeruginosa assigned as sequence type (ST) 245 for the first time in Brazil. METHODS: Antimicrobial susceptibility testing of the isolate was performed. In addition, whole-genome sequencing was performed and its virulence and resistance genes were analysed. RESULTS: The P. aeruginosa ST245 isolate was identified for the first time in Brazil in a patient with ventilator-associated pneumonia hospitalised at Hospital das Clínicas, São Paulo. Analysis of the genome showed the presence of several resistance and virulence genes. Mutations in ß-lactam resistance genes were found in ß-lactamases, outer membrane proteins, efflux pump and penicillin-binding proteins. Polymorphisms related to pathways leading to polymyxin resistance are also present, such as lipid A or keto-deoxyoctulosonate modification with aminoarabinose as well as activation of lipopolysaccharide (LPS). CONCLUSION: Such findings may represent an alert for the spread of an unusual profile in the country.

Fosfomycin in severe infections due to genetically distinct pan-drug-resistant Gram-negative microorganisms: synergy with meropenem.

Background: In vitro and clinical studies using parenteral fosfomycin have suggested the possibility of using this drug against infections caused by MDR microorganisms. The aim of this study was to describe a case series of patients treated with fosfomycin who had severe infections caused by pan-drug-resistant Gram-negative bacteria. Methods: We describe a prospective series of cases of hospitalized patients with infections caused by Gram-negative bacteria resistant to ß-lactams and colistin, treated with 16 g of fosfomycin daily for 10-14 days. Isolates were tested for antimicrobial susceptibility and synergism of fosfomycin with meropenem. We tested for resistance genes and performed typing using PCR and WGS. Results: Thirteen patients received fosfomycin (seven immunosuppressed); they had bloodstream infections (n = 11; 85%), ventilator-associated pneumonia (n = 1; 8%) and surgical site infection (n = 1; 8%), caused by Klebsiella pneumoniae (n = 9), Serratia marcescens (n = 3) and Pseudomonas aeruginosa (n = 1). Overall, eight (62%) patients were cured. Using time-kill assays, synergism between fosfomycin and meropenem occurred in 9 (82%) of 11 isolates. Typing demonstrated that K. pneumoniae were polyclonal. Eight patients (62%) had possible adverse events, but therapy was not discontinued. Conclusions: Fosfomycin may be safe and effective against infections caused by pan-drug-resistant Gram-negative microorganisms with different antimicrobial resistance mechanisms and there seems to be synergism with meropenem.

Colistin-resistant Enterobacteriaceae infections: clinical and molecular characterization and analysis of in vitro synergy.

We described 27 polyclonal colistin-resistant Enterobacteriaceae (MIC 4-16 µg/mL) infections (12 pneumonia, 12 urinary tract infection (UTI), two Bacteremia, and one skin/soft tissue infection) in which 74% harbored KPC. The isolates were polyclonal, 6 STs were identified and the colistin resistance was due to chromosome mutations. Eight patients with UTI received monotherapy, and combination therapy was given to 19 patients. Overall mortality was 37%. In vitro synergy using time-kill assay was observed in 14 of 19 (74%) isolates tested; the synergistic effect was observed for almost all isolates for the combination of three drugs: colistin, amikacin, and tigecycline. The Kaplan-Meier survival curve showed no significant difference comparing combination therapy with 2, 3, or more drugs and risk factors associated with death were dialysis and shock. These findings reinforce the fact that colistin in combination with other classes of drugs can be useful in treating infections caused by colistin-resistant CRE.