A 7-Year Brazilian National Perspective on Plasmid-Mediated Carbapenem Resistance in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex and the Impact of the Coronavirus Disease 2019 Pandemic on Their Occurrence.

BACKGROUND: Carbapenemase production is a global public health threat. Antimicrobial resistance (AMR) data analysis is critical to public health policy. Here we analyzed carbapenemase detection trends using the AMR Brazilian Surveillance Network. METHODS: Carbapenemase detection data from Brazilian hospitals included in the public laboratory information system dataset were evaluated. The detection rate (DR) was defined as carbapenemase detected by gene tested per isolate per year. The temporal trends were estimated using the Prais-Winsten regression model. The impact of COVID-19 on carbapenemase genes in Brazil was determined for the period 2015-2022. Detection pre- (October 2017 to March 2020) and post-pandemic onset (April 2020 to September 2022) was compared using the χ2 test. Analyses were performed with Stata 17.0 (StataCorp, College Station, TX). RESULTS: 83 282 blaKPC and 86 038 blaNDM were tested for all microorganisms. Enterobacterales DR for blaKPC and blaNDM was 68.6% (41 301/60 205) and 14.4% (8377/58 172), respectively. P. aeruginosa DR for blaNDM was 2.5% (313/12 528). An annual percent increase for blaNDM of 41.1% was observed, and a decrease for blaKPC of -4.0% in Enterobacterales, and an annual increase for blaNDM of 71.6% and for blaKPC of 22.2% in P. aeruginosa. From 2020 to 2022, overall increases of 65.2% for Enterobacterales, 77.7% for ABC, and 61.3% for P. aeruginosa were observed in the total isolates. CONCLUSIONS: This study shows the strengths of the AMR Brazilian Surveillance Network with robust data related to carbapenemases in Brazil and the impact of COVID-19 with a change in carbapenemase profiles with blaNDM rising over the years.

A carbapenem-resistant Acinetobacter baumannii outbreak associated with a polymyxin shortage during the COVID pandemic: an in vitro and biofilm analysis of synergy between meropenem, gentamicin and sulbactam.

BACKGROUND: During the COVID-19 pandemic, the burden of nosocomial infections caused by MDR pathogens has caused a shortage of polymyxins. Thus, we evaluated the in vitro synergism and antibiofilm activity of antimicrobial combinations and propose a test kit for synergism against carbapenem-resistant Acinetobacter baumannii (CRAB). METHODS: Fifty-six CRAB isolates were tested for synergy between meropenem, gentamicin and ampicillin/sulbactam. MICs were determined by broth microdilution. Synergism was tested using chequerboard analysis, followed by a time-kill curve. Additionally, minimum biofilm eradication concentration was determined and the antibiofilm activity of the combinations was evaluated by MTT assay and biomass reduction. A test kit was developed for routine laboratory testing to detect synergism. RESULTS: All CRAB isolates were resistant to gentamicin and ampicillin/sulbactam. Chequerboard synergism occurred against 75% of the isolates. Meropenem + ampicillin/sulbactam was the most frequent combination with synergism (69%), followed by ampicillin/sulbactam + gentamicin (64%) and meropenem + gentamicin (51%). All combinations presented only bacteriostatic activity and no bactericidal or antibiofilm effects. The routine laboratory test showed 100% accuracy compared with other in vitro assays. CONCLUSIONS: Our study demonstrates the potential role of antibiotic combinations against planktonic bacteria. In vitro synergism is possible and can be an alternative treatment for patients with CRAB infection during a polymyxin shortage.

Resolving taxonomic confusion: establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae.

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family.

Evaluation of MicroScan WalkAway for Determination of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Susceptibility in Carbapenem-Resistant Gram-Negative Bacilli.

We evaluated the performance of ceftazidime-avibactam and ceftolozane-tazobactam MicroScan Neg multidrug-resistant MIC 1 (NMR1) panel for clinical carbapenem-nonsusceptible Gram-negative bacilli isolates. We evaluated 212 clinically significant carbapenem-nonsusceptible Gram-negative bacilli (139 Pseudomonas aeruginosa and 73 KPC-producing Enterobacterales) from 71 Brazilian hospitals (2013 to 2020). Ceftazidime-avibactam and ceftolozane-tazobactam MICs from the panel were compared with a broth microdilution (BMD) test as the reference method. Essential agreement (EA) and categorical agreement (CA) were assessed. For P. aeruginosa, antimicrobial susceptibility testing error rates were calculated using the error-rate bound method. Discrepancies were initially observed with 11 isolates; 4 resolved after retesting, 2 in favor of the NMR1 and 2 in favor of the BMD method. The ceftazidime-avibactam EA (overall and evaluable) was 100% for P. aeruginosa and Enterobacterales. The CA was 100% for Enterobacterales and 98.6% for P. aeruginosa. The ceftolozane-tazobactam EA was 98.6% and 100% (overall and evaluable, respectively), and the CA was 96.4% for P. aeruginosa. For ceftazidime/avibactam, no very major error (VME) was found, and the major error (ME) rate was 4.2% (2/48). For ceftolozane-tazobactam and P. aeruginosa, using the CLSI breakpoints, the minor error (mE) was 11.4%, and no VME or ME was found. While using EUCAST breakpoints, the VME was 11.4% with no ME. The mE becomes ME or VME in the absence of the intermediate category. All categorical errors were also within 1 log of MIC variation, and the adjusted error rate for CLSI/EUCAST was 0% (0/212). The NMR1 panel is an option to test ceftazidime-avibactam for KPC-producing Enterobacterales and carbapenem-nonsusceptible P. aeruginosa. When a MIC of 4 mg/liter for ceftolozane-tazobactam is obtained using this method, an alert could be created, and the results could be confirmed by an alternative method.

Emended description of the genus Phytobacter, its type species Phytobacter diazotrophicus (Zhang 2008) and description of Phytobacter ursingii sp. nov.

The species Phytobacter diazotrophicus and the associated genus Phytobacter were originally described by Zhanget al. [Arch Microbiol189 (2008), 431-439] on the basis of few endophytic nitrogen-fixing bacteria isolated from wild rice (Oryza rufipogon) in China. In this study, we demonstrate that a number of clinical isolates that were either described in the literature, preserved in culture collections, or obtained during a 2013 multi-state sepsis outbreak in Brazil also belong to the same genus. 16S rRNA gene sequencing, multilocus sequence analysis based on gyrB, rpoB, atpD and infB genes, as well as digital DNA-DNA hybridization support the existence of a second species within the genus Phytobacter. All isolates from the recent Brazilian outbreak, along with some older American clinical strains, were found to belong to the already described species Phytobacterdiazotrophicus, whereas three clinical strains retrieved in the USA over a time span of almost four decades, could be assigned to a new Phytobacter species. Implementation of an extended set of biochemical tests showed that the two Phytobacter species could phenotypically be discriminated from each other by the ability to utilize l-sorbose and d-serine. This feature was limited to the strains of the novel species described herein, for which the name Phytobacter ursingii sp. nov. is proposed, with ATCC 27989T (=CNCTC 5729T) as the designated type strain. An emended description of the species Phytobacter diazotrophicus and of the genus Phytobacter is also provided.

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

Activity of antimicrobial combinations against KPC-2-producing Klebsiella pneumoniae in a rat model and time-kill assay.

This study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae infection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P = 0.018) and was antagonistic. In vivo and in vitro studies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain.

Phenotypic and molecular characterization of 942 carbapenem-resistant Enterobacteriaceae (CRE) in southern Brazil.

Carbapenem-resistant Enterobacteriaceae (CRE) is a major international health problem, and its identification in developing countries is based exclusively on phenotypic methods. The aim of this study was to assess the sensitivity and related parameters of the modified Hodge test (MHT). The assessment was performed in a large number of isolates obtained from different hospitals in several cities of a south Brazilian state. Bacterial species were identified using an automated method. The MHT was performed according to the guidelines set by the CLSI. The gene blaKPC was amplified in order to confirmation CRE expression. The sensitivity, specificity, positive, and negative predictive values were calculated. A total of 942 isolates were submitted to the reference laboratory for confirmation; 143 showed a negative MHT (15.18%) result, while 784 were positive (83.23%), and 15 samples displayed an indeterminate MHT (1.59%) result. All samples expressed the KPC-2 enzyme. Sensitivity, specificity, positive, and negative predictive percentiles were 99%, 89%, 98%, and 99% respectively. We conclude that the modified Hodge test is a reliable test for the prediction of KPC-producing bacteria.

First report of NDM-1-producing Acinetobacter baumannii sequence type 25 in Brazil.

New Delhi metallo-ß-lactamase 1 (NDM-1) was first identified in Brazil in Enterobacter hormaechei and Providencia rettgeri in 2013. Here, we describe the first case of NDM-1-producing Acinetobacter baumannii sequence type 25 isolated from the urinary tract of a 71-year-old man who died of multiple complications, including A. baumannii infection. The NDM-1 gene was detected by quantitative PCR, and its sequence confirmed its presence in an ∼ 100-kb plasmid.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Oral doxycycline to carbapenem-resistant Acinetobacter baumannii infection as a polymyxin-sparing strategy: results from a retrospective cohort.

Acinetobacter baumannii infection presents a high mortality rate and few therapeutic options. This study aimed to evaluate clinical-microbiological characteristics and prognosis factors of patients diagnosed with A. baumanni. infections treated with oral doxycycline. A retrospective cohort of hospitalized patients with confirmed Acinetobacter spp. infection between 2018 and 2020 receives at least 3 days of oral doxycycline. Clinical and microbiological data were evaluated, including the outcome and molecular characterization of A. baumannii. Doxycycline minimal inhibitory concentrations were evaluated by the broth dilution method. One hundred patients were included with a median age of 51 years. The leading site of infection was pulmonary (n = 62), followed by the soft tissues and skin (n = 28). A. baumannii resistant to carbapenem was found on 94%. The gene blaOXA-23 and blaOXA-51 were amplified in all recovered isolates of A. baumannii (n = 44). Doxycycline MIC50 and MIC90 were 1 µg/mL and 2 µg/mL, respectively. Death rate at 14 days and 28 days of follow-up was 9% and 14%, respectively. The prognostic factors related to death at end of follow-up were age > 49 years [85.7% vs. 46%, CI 95% 6.9 (1.4-32.6), P = 0.015] and hemodialysis [28.6% vs. 7%, CI 95% 5.33 (1.2-22.1), P = 0.021]. Patients treated with doxycycline to A. baumannii presented a relatively low death rate, and risk factors related to death were age and hemodialysis. Further and larger studies should compare polymyxin to doxycycline to better understand the differences between these therapeutic options.

Dissemination of NDM-producing bacteria in Southern Brazil.

BACKGROUND: The dissemination of NDM-1 carbapenemases (New Delhi Metallo-ß-lactamase) is a global public health problem, mainly in developing countries. The aim of this study was to characterize the spread of NDM-producing bacteria in the Southern Brazilian states analyzing epidemiological, molecular, and antimicrobial susceptibility aspects. METHODS: A total of 10,684 carbapenem-resistant isolates of Enterobacterales, Pseudomonas spp. and Acinetobacter spp. obtained from several hospitals in eight cities in Southern Brazil were screened, and 486 NDM-producing bacteria were selected. RESULTS: The incidence varied from 0.5 to 77 cases/100.000 habitants. ST11, ST15, ST340 and ST674 were the most common in K. pneumoniae. A total of 5 plasmids were identified in one K. pneumoniae strain: Col440I, Col440II, IncFIA(HI1), IncFIB(K), IncFIB(pQil)/ IncFII(K), and IncR. CONCLUSIONS: The number of patients with NDM-producing bacteria has increased in Southern Brazil, whose gene is present in different plasmids, explaining the expansion of this enzyme.

Distribution of genes encoding 16S rRNA methyltransferase in plazomicin-nonsusceptible carbapenemase-producing Enterobacterales in Brazil.

BACKGROUND: The presence of 16S rRNA methyltranferases (16S-RMTases) in carbapenemase-producing Enterobacterales (CPE) is a major concern because it inactivates all clinical use of aminoglycosides, including plazomicin. The aim of this study is to investigate the prevalence of 16S-RMTases in CPE nonsusceptible to plazomicin collected in different Brazilian hospitals. METHODS: All isolates with plazomicin MIC ≥ 4 µg/mL (n = 67) were screened for the presence of 16S-RMTases by sequencing. RESULTS: 54 (80.6%) isolates encoded 16S-RMTase genes (41 rmtB1, 7 armA, 3 rmtD2, 1 rmtD1 and 2 rmtC). Among 41 samples rmtB1 positive, 40 co-harbored blaKPC-2 and 1 blaOXA-48 gene. Of the seven isolates harboring armA gene, 6 were New Delhi Metallo-beta-lactamase (NDM)-producer. rmtD was only found in isolates Klebsiella pneumoniae Carbapenemase (KPC)-producers, one in Serratia marcescens with rmtD2, not reported in Brazil. CONCLUSION: The co-existence of 16S-RMTase and CPE is worrisome because of limited treatment options and the endemic characteristic of (KPC) and NDM in Brazil.

Elution methods to evaluate colistin susceptibility of Gram-negative rods.

Recently it was developed the Colistin Broth Disk Elution test which uses colistin disks as a source of these antibiotics. The aim of this study was to evaluate the performance of protocols that used diminished volumes of the reagents: the Colistin Broth Microelution (CBM) (1 mL) and the Microelution-Plates Test (MPT) (200 µL), as well as the Colistin Susceptibility Test Tube (CSTT), which uses only one colistin disk added to a tube containing broth. The tests were performed with 85 Gram-negative isolates collected from surveillance studies. The CBM, MPT, and CSTT tests presented a good Categorical Agreement (CA), Essential Agreement (EA), sensitivity and specificity to Enterobacterales isolates, however the ME and VME were less satisfactory. The results for non-fermentative isolates were not satisfactory. In conclusion, the proposed methods, mainly the CSTT, can be used as screening tests to detect colistin resistant among Enterobacterales, as they are an easy and inexpensive option to the reference method.

Validation of multiplex PCR for the diagnosis of acute bacterial meningitis in culture negative cerebrospinal fluid.

INTRODUCTION: This study evaluated the operational characteristics of the multiplex polymerase chain reaction (PCR) for cerebrospinal fluid (CSF) from patients with cellular and biochemical characteristics of acute bacterial meningitis and positive or negative CSF cultures. METHODS: Multiplex PCR was performed for 36 CSF samples: culture-proven acute bacterial meningitis (n = 7), culture-negative acute bacterial meningitis (n = 17), lymphocytic meningitis (n = 8), and normal CSF (n = 4). The operational characteristics of multiplex PCR were evaluated with definite and probable bacterial meningitis, using culture positive, cytological and biochemical CSF characteristics as the gold standard. RESULTS: Multiplex PCR for CSF was efficient in the group with CSF cellular and biochemical characteristics of acute bacterial meningitis but with a negative CSF culture. This group demonstrated high specificity, positive predictive value, and efficiency. CONCLUSIONS: Multiplex PCR for CSF can improve the speed and accuracy of acute bacterial meningitis diagnosis in a clinical setting as a complement to classical immunological and bacteriological assays in CSF. It is also useful for CSF culture-negative acute bacterial meningitis.

Low level of polymyxin resistance among nonclonal mcr-1-positive Escherichia coli from human sources in Brazil.

We report 26 human isolates of mcr-1-positive Escherichia coli, most of them (65.4%) with a polymyxin B MIC of 2 mg/L. Seventeen out of the 24 mcr-1-positive E. coli proved to be nonclonal by rep-PCR which strengthens the hypothesis of environmental or animal origin of these strains and reinforces the one health context of antimicrobial resistance.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates

ABSTRACT Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.