Detection of polymyxins resistance among Enterobacterales: evaluation of available methods and proposal of a new rapid and feasible methodology.

BACKGROUND: Fast and accurate detection of polymyxins resistance is necessary as they remain the last resources to treat infections caused by Carbapenem-resistant Enterobacterales in many regions. We evaluated the rapid colorimetric polymyxin B elution (RCPE) and developed its miniaturized version, RCPE microelution (RCPEm), aiming to detect polymyxins resistance among Enterobacterales. METHODS: The methodologies consist of exposing the bacterial population in a solution (NP solution) where polymyxin B disks were previously eluted to obtain a concentration of 2 µg/mL for RCPE and 3 µg/mL for RCPEm. RESULTS: Two hundred sixty-seven Enterobacterales were evaluated, 90 (33.7%) resistant to polymyxin B by broth microdilution. It was observed 0.6% of major error (ME) by RCPE, with a specificity of 99.4%. The miniaturized version (RCPEm) presented the same ME and specificity values, but slightly higher sensitivity (97.8% vs. 95.6%) with 2.2% of very major error (VME). CONCLUSIONS: RCPE and RCPEm proved to be useful alternatives to determine polymyxin B susceptibility in clinical microbiology laboratories, presenting low cost, being easy to perform, and demanding short incubation time.

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene.

Variants of concern (VOCs) of SARS-CoV-2 are viral strains that have mutations associated with increased transmissibility and/or increased virulence, and their main mutations are in the receptor binding domain (RBD) region of the viral spike. This study aimed to characterize SARS-CoV-2 VOCs via Sanger sequencing of the RBD region and compare the results with data obtained via whole genome sequencing (WGS). Clinical samples (oro/nasopharyngeal) with positive RT-qPCR results for SARS-CoV-2 were used in this study. The viral RNA from SARS-CoV-2 was extracted and a PCR fragment of 1006 base pairs was submitted for Sanger sequencing. The results of the Sanger sequencing were compared to the lineage assigned by WGS using next-generation sequencing (NGS) techniques. A total of 37 specimens were sequenced via WGS, and classified as: VOC gamma (8); delta (7); omicron (10), with 3 omicron specimens classified as the BQ.1 subvariant and 12 specimens classified as non-VOC variants. The results of the partial Sanger sequencing presented as 100% in agreement with the WGS. The Sanger protocol made it possible to characterize the main SARS-CoV-2 VOCs currently circulating in Brazil through partial Sanger sequencing of the RBD region of the viral spike. Therefore, the sequencing of the RBD region is a fast and cost-effective laboratory tool for clinical and epidemiological use in the genomic surveillance of SARS-CoV-2.

Direct Detection of KPC Peak from Positive Blood Cultures Using MALDI-TOF MS: Are We There Yet?

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 blaKPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 blaKPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a "buffer" zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.

Evaluation of Early Reading of Broth Microdilution Technique for Polymyxin B.

Delay in the results of standard phenotypic susceptibility tests is the main obstacle to adequate antibiotic treatment. For this reason, the European Committee for Antimicrobial Susceptibility Testing has proposed the Rapid Antimicrobial Susceptibility Testing for the disk diffusion method directly from blood culture. However, to date, there are no studies evaluating early readings of polymyxin B broth microdilution (BMD), the only standardized methodology for assessing susceptibility to polymyxins. This study aimed to evaluate modifications in the BMD technique for polymyxin B using fewer antibiotic dilutions and reading after an incubation time of 8-9 hr (early reading) in comparison to 16-20 hr of incubation (standard reading) for isolates of Enterobacterales, Acinetobacter baumannii complex, and Pseudomonas aeruginosa. A total of 192 isolates of gram-negative bacteria were evaluated and the minimum inhibitory concentrations were read after early and standard incubations. The early reading presented 93.2% of essential agreement and 97.9% of categorical agreement with the standard reading of BMD. Only three isolates (2.2%) presented major errors and only one (1.7%) presented a very major error. These results indicate a high agreement between the early and the standard reading times of BMD of polymyxin B.

Evaluation of filter paper to transport oro/nasopharyngeal samples to detect SARS-CoV-2 by RT-qPCR.

PURPOSE: To evaluate filter paper as a means to transport oro/nasopharyngeal samples from laboratories with few resources for SARS-CoV-2 detection by RT-qPCR in a central laboratory that usually performs this technique as routine. METHODS: A total of 40 specimens were evaluated in parallel by RT-qPCR carried out after RNA extraction using two different protocols: direct RNA extraction (Protocol A - reference method) and RNA extraction after impregnation in filter paper (Protocol B). RESULTS: The RT-qPCR for SARS-CoV-2 using Protocol B presented 97.22% (35/36) of agreement for SARS-CoV-2-positive samples when compared to the reference method (Protocol A), even for specimens with low viral load (increased Ct values). Noteworthy, three clinical specimens which were categorized as inconclusive by Protocol A presented amplification of both N1 and N2 targets using Protocol B, presenting positive results for SARS-CoV-2. CONCLUSION: The use of filter paper to transport oro/nasopharyngeal clinical samples presented very satisfactory results to detect SARS-CoV-2 by RT-qPCR. In addition, it proved to be a feasible and sensitive approach, being able to generate the detection of SARS-CoV-2 even at low concentrations, without presenting false-negative results.

Evaluation of Clinical Course of Gamma (P.1) Variant of Concern versus Lineages in Hospitalized Patients with COVID-19 in a Reference Center in Brazil.

The SARS-CoV-2 variant of concern (VOC) gamma (P.1) has increased transmissibility and resulted in elevated hospitalization and mortality rates in Brazil. We investigated the clinical course of COVID-19 caused by gamma and non-VOCs at a reference hospital in Brazil in a retrospective cohort study with nonelderly hospitalized patients from two periods, before and after the emergence of gamma. Cohort 1 included patients from both periods whose samples would be eligible for whole-genome sequencing (WGS). Cohort 2 was composed of randomly selected patients from Cohort 1 whose samples were submitted to WGS. A total of 433 patients composed Cohort 1: 259 from the first and 174 from the second period. Baseline characteristics were similar, except for a higher incidence of severe distress respiratory syndrome at admission in patients from the second period. Patients from the second period had significantly higher incidence rates of advanced respiratory support (adjusted hazard ratio [aHR]: 2.04; 95% confidence interval [CI], 1.60-2.59), invasive ventilatory support (aHR: 2.72; 95% CI: 2.05-3.62), and 28-day mortality from the onset of symptoms (aHR: 2.62; 95% CI: 1.46-4.72). A total of 86 (43 gamma and 43 non-gamma) patients composed Cohort 2. Patients with confirmed gamma VOC infections had higher advanced ventilatory support and mortality rates than non-gamma-infected patients. Our study suggests that non-elderly patients hospitalized for COVID-19 in the second period (used as a proxy of gamma infection) had a more severe clinical course. This might have contributed to higher hospitalization and death rates observed in the second wave in Brazil.

MALDI-TOF mass spectrometry for direct KPC detection among Enterobacterales.

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller-Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643-28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

The occurrence of antimicrobial residues and antimicrobial resistance genes in urban drinking water and sewage in Southern Brazil.

Antimicrobial resistance (AMR) is currently discussed as an important issue worldwide, and the presence of antimicrobial residues (ARs) and antimicrobial resistance genes (ARGs) in the environment, especially in the water sources, is a challenge for public health. This study was conducted to evaluate the occurrence and diversity of AR and ARG in water sources from an urban center, in Southern Brazil. A total of thirty-two water samples from drinking water treatment plants (24) and sewage systems (8) were collected during two annual samplings, winter and summer. The PCR was performed by 18 ARGs, and the detection of 47 ARs was performed by LC-MS/MS. All sewage samples presented carbapenemases, ESBL, and mcr-1 genes as well as quinolones and sulfamethoxazole residues. In drinking water, we just detected blaTEM and tetB genes and doxycycline residues in samples before treatment. This study provides data about AR and ARG in drinking water and sewage systems showing that these sources are important reservoirs of both. The limited effectiveness of wastewater treatment processes to remove mainly AR demonstrates the need to implement better protocols of disinfection, in order to limit the spread of AMR in the environment.

Genomic Surveillance of SARS-CoV-2 Lineages Indicates Early Circulation of P.1 (Gamma) Variant of Concern in Southern Brazil.

The SARS-CoV-2 P.1 lineage emerged in Amazonas (AM), North Brazil and its evolution has been dynamically reported associated with increased transmissibility and/or immune evasion. Here, we evaluated the lineages circulating in 29 cities in Rio Grande do Sul (RS), Southern Brazil between March 2020 and May 2021 and investigated the genetic events associated with the emergence of the P.1. A total of 202 oro/nasopharyngeal SARS-CoV-2 specimens from patients during routine hospital care were submitted to whole-genome sequencing. Phylogenetic and Bayesian Evolutionary Analyses of the P.1 lineage were carried out to determine the relationship between sequences from RS and AM and dated their common ancestor and origin. One hundred six (53%) sequences were assigned as P.1 and most carried the 22 lineage-defining mutations. All the P.1 sequences included other important mutations, such as P314L and R203K/G204R, and revealed a high genetic diversity in the phylogenetic tree. The time-scaled inference suggests that the oldest P.1 sequences from different Brazilian states share a ancestor with those from AM, but the origin of some sequences from RS is unknown. Further, the common ancestor of sequences from RS is dated to mid-June/July 2020, earlier than those previously reported from AM. Our results demonstrate that there is a high degree of genetic diversity among P.1 sequences, which suggests a continuous evolution and community spread of the virus. Although the first P.1 outbreak was reported in AM, the lineage was associated with multiple introductory events and had already been circulating in Southern Brazil prior to November 2020. IMPORTANCE The SARS-CoV-2 P.1 lineage is associated with increased transmissibility and/or immune evasion and presents a dynamic evolution in Brazil. The significance of our research relies in the fact that we evaluated the SARS-CoV-2 lineages circulating in Southern Brazil between March 2020 and May 2021. This evaluation allowed us to detect the genetic events associated with the emergence of the P.1 and its sublineages. This study is important because we were able to establish that the common ancestor of P.1 sequences from Rio Grande do Sul, Southern Brazil, is dated of mid-June/July 2020, earlier than the P.1 sequences previously reported from Amazonas (AM) state. Noteworthy, the high degree of genetic diversity among P.1 sequences found in this study suggests a continuous evolution and community spread of the virus. Moreover, the oldest P.1 sequences from different Brazilian states share a ancestor with those from AM.

Evaluation of a rapid susceptibility test of polymyxin B by MALDI-TOF.

Introduction: Infections caused by multidrug-resistant microorganisms have become increasingly common in hospital environments around the world. Gram-negative bacilli stands out among multidrug-resistant bacteria mostly due to the production of carbapenemase enzymes which lead to resistance to most ß-lactam antibiotics including the carbapenems. As a consequence, polymyxins have been reintroduced in the clinic as a last resort to treat infections caused by Gram-negative bacilli resistant to carbapenems. However, the only reliable method to evaluate the susceptibility to polymyxins is the broth microdilution, a laborious and time-consuming technique. Among infections caused by multidrug-resistant bacteria, bloodstream infections are the most worrisome as they can lead to sepsis and septic shock with high mortality rates. Objective: Considering the severity of sepsis and the need for a treatment guided for the susceptibility test in vitro, this work aimed to evaluate a rapid method of polymyxins susceptibility either from colonies grown on agar or directly from positive blood culture bottles using the technology of MALDI-TOF. Methods: The method was based on the "direct on target microdroplets growth assay" (DOT-MGA) originally developed by Idelevich and collaborators with some modifications (Adapted DOT-MGA). Isolates of Enterobacterales and non-fermenting Gram-negative bacilli resistant to carbapenems were obtained from patients attending a tertiary care hospital in southern Brazil and tested as follows: 122 isolates from colonies grown on agar plates and 117 isolates directly from spiked positive blood cultures. Results: The adapted DOT-MGA presented 95 and 100% of categorical agreement considering the colonies grown on agar plates and directly from positive blood cultures, respectively. Discussion: The adapted DOT-MGA test proved to be a reliable technique to evaluate the susceptibility to polymyxins to be used in microbiology laboratories with the MALDI-TOF equipment.

RT-qPCR half-reaction optimization for the detection of SARS-CoV-2.

INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

Characterization of an emergent high-risk KPC-producing Klebsiella pneumoniae lineage causing a fatal wound infection after spine surgery.

Surgical site infections in instrumented posterior lumbar interbody fusion surgery are normally due to gram-positive bacteria, but gram-negative bacteria can cause infections in cases involving lower lumbar interventions as its closer to the perianal area. Here we report an uncommon fatal wound infection caused by a multidrug-resistant Klebsiella pneumoniae after an elective spine surgery. In silico analysis revealed that LWI_ST16 belonged to ST16, an emergent international clone notable for its increased virulence potential. We also observed that this strain carried a conjugative IncF plasmid encoding resistance genes to beta-lactams (blaKPC-2 and blaOXA-1), tetracycline (tetA), aminoglycosides and fluoroquinolones (aac(6')-Ib-cr). The carbapenemase encoding gene blaKPC-2 was located on a Tn4401e transposon previously characterized to increase blaKPC expression. LWI_ST16 is a strong biofilm producer on polystyrene and capable of forming tower-like structures on a titanium device like the one inserted in the patient's spine. Our findings strengthen the valuable contribution of continuous surveillance of multidrug-resistant and high-risk K. pneumoniae clones to avoid unfavourable clinical outcomes.

Evaluation of filter paper to transport inactivated bacteria to detect carbapenem resistance genes by multiplex real-time PCR using high-resolution melting.

Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.

SARS-CoV-2 nucleic acid shedding is variable for every person.

INTRODUCTION: Considering the persistent positivity on RT-qPCR tests, the results of SARS-CoV-2 were monitored to evaluate the viral RNA shedding period. METHODS: Between March and June 2020, the sequential results of 29 healthcare workers' were monitored using RT-qPCR. RESULTS: More than 50% of the individuals remained RT-qPCR positive after 14 days. Furthermore, this is the first study to describe positive RT-qPCR for SARS-CoV-2 in a healthcare worker with mild symptoms 95 days after the first positive test. CONCLUSIONS: Sequential RT-qPCR results were heterogeneous, and the viral RNA shedding period is unique for each person.

NDM-1-encoding plasmid in Acinetobacter chengduensis isolated from coastal water.

BACKGROUND: Acinetobacter spp. may cause difficult-to-treat nosocomial infections due to acquisition of carbapenemases, including New Delhi metallo-ß-lactamase (NDM). This genus has been pointed out as a possible actor in the early dissemination of blaNDM, and this gene has been documented in a variety of species. OBJECTIVE: Here we describe an Acinetobacter chengduensis (isolate FL51) carrying blaNDM recovered from coastal water in Brazil. METHODS: In vitro techniques included antimicrobial susceptibility and minimum inhibitory concentration tests, PCR, plasmid profile and matting-out/transformation assays. In silico approaches comprised comparative genomic analyses using appropriate databases. RESULTS: FL51 grew at room temperature in a variety of culture media, excluding MacConkey. It showed resistance to all beta-lactams tested and to ciprofloxacin. blaNDM-1 was identified, and a single replicon was observed in plasmid profile. In silico DNA hybridization revealed Acinetobacter FL51 as being Acinetobacter chengduensis. blaNDM-1 was flanked upstream by ISAba14-aphA6-ISAba125 and downstream by bleMBL-trpF-Δtat, inserted in a 41,068 bp non typeable plasmid named pNDM-FL51. This replicon showed high coverage and identity with other sequences present in plasmids deposited on the GenBank database, recovered almost exclusively from Acinetobacter spp., associated with hospital settings and animal sources. CONCLUSION: We described a recently described environmental Acinetobacter species carrying a plasmid-borne blaNDM associated with a Tn125-like structure. Our findings suggest that replicon may play an important role in blaNDM dissemination among distinct settings within this genus and may support the theory of blaNDM emergence from an environmental Acinetobacter.

Detection of SARS-CoV-2 lineage P.1 in patients from a region with exponentially increasing hospitalisation rate, February 2021, Rio Grande do Sul, Southern Brazil.

The emergence of SARS-CoV-2 P.1 lineage coincided with a surge in hospitalisations in the North region of Brazil. In the South region's Rio Grande do Sul state, severe COVID-19 case numbers rose 3.8 fold in February 2021. During that month, at a COVID-19 referral hospital in this state, whole-genome sequencing of a subset of cases' specimens (n = 27) revealed P.1 lineage SARS-CoV-2 in most (n = 24). Findings raise concerns regarding a possible association between lineage P.1 and rapid case and hospitalisation increases.

Increased frequency of blaNDM in a tertiary care hospital in southern Brazil.

Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The blaKPC-like was identified in 3003 isolates (85.8%) and the blaNDM-like was the second most common gene (351 isolates-10%). There was a steady increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020. The increase of blaNDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the blaNDM.