Challenges in the Detection of Polymyxin Resistance: From Today to the Future.

Antimicrobial resistance is known to be one of the greatest global threats to human health, and is one of the main causes of death worldwide. In this scenario, polymyxins are last-resort antibiotics to treat infections caused by multidrug-resistant bacteria. Currently, the reference test to evaluate the susceptibility of isolates to polymyxins is the broth microdilution method; however, this technique has numerous complications and challenges for use in laboratory routines. Several phenotypic methods have been reported as being promising for implementation in routine diagnostics, including the BMD commercial test, rapid polymyxin NP test, polymyxin elution test, culture medium with polymyxins, and the Polymyxin Drop Test, which require materials for use in routines and must be easy to perform. Furthermore, Sensititre®, molecular tests, MALDI-TOF MS, and Raman spectroscopy present reliable results, but the equipment is not found in most microbiology laboratories. In this context, this review discusses the main laboratory methodologies that allow the detection of resistance to polymyxins, elucidating the challenges and perspectives.

Conciliação medicamentosa em hospital universitário: estratégia de segurança em pacientes neurocirúrgicos / Medication reconciliation in a university hospital: safety strategy in neurosurgical patients / Conciliación de medicamentos en un hospital universitario: estrategia de seguridad en pacientes neuroquirúrgicos

Esse trabalho teve como objetivo identificar as discrepâncias medicamentosas, por meio do serviço de conciliação de medicamentosa, em pacientes admitidos na clínica cirúrgica de uma unidade especializada no atendimento de doença relacionadas ao sistema neuromuscular na cidade de Manaus, Amazonas. Trata-se de um estudo descritivo e prospectivo, realizado no período de setembro a dezembro de 2020 em pacientes submetidos a conciliação medicamentosa. Na primeira etapa realizou-se a anamnese farmacêutica em formulário semiestruturado e foi elaborada a melhor história possível de medicamentos (MHPM). Os medicamentos prescritos na admissão foram comparados com a MHPM e as discrepâncias foram identificadas e classificadas quanto a intencionalidade e tipo. Ao total 54 pacientes foram incluídos no estudo, sendo que para 32 foi realizada a conciliação medicamentosa por usarem medicamentos de uso contínuo. Foram identificadas 20 discrepâncias intencionais, 3 discrepâncias intencionais não documentadas e 12 discrepâncias não intencionais. Omissão de medicamentos foi o tipo de discrepância mais comum (86%). Diante do exposto, concluímos que a conciliação medicamentosa mostrou-se um importante recurso para identificação de discrepâncias na transição de cuidado de pacientes com doenças neurológicas, principalmente no que se refere à omissão de medicamentos. As intervenções farmacêuticas a partir das discrepâncias encontradas, conseguiram mitigar erros de medicação e possíveis eventos adversos, aumentando a segurança do paciente.

This work aimed to identify medication discrepancies, through the medi- cation reconciliation service, in patients admitted to the surgical clinic of a unit special- ized in treating diseases related to the neuromuscular system in the city of Manaus, Ama- zonas. This is a descriptive and prospective study, carried out from September to Decem- ber 2020 in patients undergoing medication reconciliation. In the first stage, the pharma- ceutical anamnesis was carried out in a semi-structured form and the best possible medi- cation history (MHPM) was prepared. Medications prescribed on admission were com- pared with the MHPM and discrepancies were identified and classified according to in- tentionality and type. 54 patients were included in the study, and for 32 medication rec- onciliation was performed because they used continuous medication. A total of 20 inten- tional discrepancies, 3 intentional undocumented discrepancies and 12 unintentional dis- crepancies were identified. Medication omission was the most common type of discrep- ancy (86%). We conclude that medication reconciliation proved to be an important re- source for identifying discrepancies in the transition of care for patients with neurological diseases, especially with regard to medication omission. Pharmaceutical interventions, based on the discrepancies found, managed to mitigate medication errors and possible adverse events, increasing patient safety.

Este estudio tuvo como objetivo identificar las discrepancias de medicación a través del servicio de reconciliación de medicamentos, en pacientes internados en la clínica quirúrgica de una unidad especializada en el tratamiento de enfermedades relacio- nadas con el sistema neuromuscular en la ciudad de Manaus, Amazonas. Este es un estu- dio descriptivo y prospectivo, realizado de septiembre a diciembre de 2020 en pacientes en conciliación de medicación. En la primera etapa se realizó la anamnesis farmacéutica de forma semiestructurada y se elaboró el mejor historial de medicación posible (MHPM). Los medicamentos prescritos al ingreso se compararon con el MHPM y se identificaron las discrepancias y se clasificaron según la intencionalidad y el tipo. Un total de 54 pacientes fueron incluidos en el estudio, y a 32 se les realizó conciliación de medi- cación por utilizar medicación continua. Se identificaron un total de 20 discrepancias in- tencionales, 3 discrepancias intencionales no documentadas y 12 discrepancias no inten- cionales. La omisión de medicamentos fue el tipo más común de discrepancia (86%). Concluimos que la conciliación de medicamentos demostró ser un recurso importante para identificar discrepancias en la transición de la atención a pacientes con enfermedades neurológicas, especialmente en lo que respecta a la omisión de medicamentos. Las inter- venciones farmacéuticas, en base a las discrepancias encontradas, lograron mitigar errores de medicación y posibles eventos adversos, aumentando la seguridad del paciente PALABRAS CLAVE: Conciliación de Medicamentos; Seguridad del Paciente; Servicio de Farmacia Hospitalaria; Neurología.

Evaluation of the susceptibility test of polymyxin B using the commercial test Policimbac®.

Broth microdilution (BMD), the reference method to determine bacterial susceptibility to polymyxins, is a laborious and time-consuming technique. Policimbac® is a commercial test panel which uses lyophilized polymyxin B to determine the minimum inhibitory concentration for Gram-negative isolates. This study evaluated the performance of Policimbac® in comparison with BMD for 110 isolates. Although the Policimbac® presented a very low essential agreement, the categorical agreement with BMD was optimal. Policimbac® is an alternative approach to BMD for evaluating the susceptibility to polymyxin B.

Evaluation of EDTA and Dipicolinic Acid in Broth Microdilution with Polymyxin B as a Phenotypic Test to Detect the mcr-1 Gene.

Polymyxins (colistin and polymyxin B) have recently regained significant importance as last-line drugs to treat infectious diseases due to multidrug-resistant gram-negative bacteria. However, resistance to polymyxins has increased, and the recognition of plasmid-mediated resistance (by the mcr gene) has led to an epidemiological concern. We aimed to evaluate the reduction of the polymyxin B minimum inhibitory concentration (MIC) in the presence of EDTA or dipicolinic acid (DPA) by using the broth microdilution (BMD) method for phenotypic screening of acquired polymyxin resistance mediated by the mcr-1 gene. Overall, 94 Enterobacterales (48 polymyxin-resistant and 46 polymyxin-susceptible) were evaluated: 47 mcr-1 positive (36 Escherichia coli, 2 Klebsiella pneumoniae, and 9 Salmonella spp.) and 47 mcr-1 negative (3 E. coli and 44 K. pneumoniae-27 isolates with MIC from ≤0.125 to 8 µg/mL and 20 isolates with MIC from 16 to 64 µg/mL). Results were categorized as positive when the chelator decreased the original BMD MIC by ≥2 logs. The majority (95.7%) of mcr-1 positive isolates displayed at least a 3 log dilution decrease in the MIC of polymyxin B with EDTA or DPA. The EDTA-based BMD assay detected 45 mcr-1-positive isolates, with only one false-positive among the mcr-1-negative isolates (sensitivity [SN], 95.7%; specificity [SP], 97.9%), whereas the DPA-based BMD assay detected 44 mcr-1-positive isolates (SN, 93.6%; SP, 95.7%), with two false-positive results. The accuracy of EDTA- and DPA-based BMD assays were 97% and 95%, respectively. The EDTA- and DPA-based assays were demonstrated to be reliable methods to detect mcr-1 positive isolates with excellent accuracy.

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.

Rapid detection of the main carbapenemases in Brazil directly from spiked blood culture using the RESIST-3 O.K.N. immunoassay.

The emergence of carbapenem-resistant Enterobacterales (CRE) is a matter of public health concern. Carbapenemases are the main mechanism of resistance among CRE, and its rapid detection is essential. The detection of carbapenemases usually requires culture-based methods and molecular assays, which may be costly and need long turnaround times. Recently, an easy and rapid immunochromatographic assay for carbapenemases (OXA-48, KPC, and NDM) detection based in lateral flow immunoassay with specific monoclonal antibodies on a nitrocellulose membrane has been developed. We aimed to evaluate the RESIST-3 O.K.N. in colonies from pure culture as well as in spiked blood cultures with Enterobacterales. All carbapenemase producers (CP) presenting the OXA-48-like, KPC, and NDM enzymes presented positive results in both pure colonies and spiked blood cultures. None of the carbapenemase non-producers (CNP) presented positive results in the tests. A total of 97% CP isolates presented positive results in pure colonies in less than 5 min. For CP directly from blood culture, the mean time to positivity for OXA-48-like and KPC was 1 min, whereas it was 25 min for NDM. Our results indicate that this immunoassay can be used to detect carbapenemases directly from blood culture bottles in a routine diagnostic laboratory, which would reduce the turnaround time of CP detection.

Detection of Enterobacterales resistant to polymyxins using Rapid Polymyxins NP test.

Two hundred isolates of Enterobacterales were tested by Rapid Polymyxins NP for the detection of polymyxin resistance and compared to the reference test broth microdilution (BMD). The sensitivity and specificity of the NP test were 98% and the results are faster than the BMD, decreasing from approximately 24 to 2 h.

Rapid Detection of Carbapenemase Production Directly from Blood Culture by Colorimetric Methods: Evaluation in a Routine Microbiology Laboratory.

The aim of this study was to evaluate the two rapid colorimetric methods (CNPt-Direct and Blue-Carba) for the detection of carbapenemase production directly from blood culture in a routine microbiology laboratory. The methods were initially evaluated on spiked blood cultures with 61 carbapenemase-positive isolates. Afterwards, they were used in blood cultures (314 samples were evaluated) obtained from patients in a routine microbiology laboratory during a period of 6 months. The colorimetric methods were compared to the conventional culture of blood. The results of the spiked blood cultures indicated that both colorimetric methods presented positive results for the vast majority (95%) of the isolates harboring KPC, NDM, and IMP genes. However, the assay failed to detect many GES- and OXA-48-like-positive isolates (65% positive results). In the second part of the study, a total of 314 blood cultures from patients were evaluated, and 33 yielded Enterobacteriaceae isolates resistant to meropenem (30 isolates were positive for carbapenemases according to PCR). The colorimetric tests correctly detected 24 out of the 30 carbapenemase-positive isolates directly from the blood vial (80% positive results). Overall positive percent agreement and negative percent agreement were 80% and 100%, respectively. The colorimetric assays are simple and cost-effective methods that can be implemented in a routine microbiology laboratory, diminishing the time necessary to detect carbapenemase-producing isolates from 24 to 48 h to 3 to 5 h. Moreover, according to our results, the positive colorimetric test results do not need to be confirmed and can be immediately provided to the attending physician.

Antimicrobial activity of plazomicin against Enterobacteriaceae-producing carbapenemases from 50 Brazilian medical centers.

Plazomicin is a next-generation aminoglycoside with activity against Enterobacteriaceae, including carbapenemase-producing Enterobacteriaceae (CPE). The aim of this study was to evaluate the activity of plazomicin against CPE (Klebsiella spp., Escherichia coli, Serratia spp., Enterobacter spp., Citrobacter spp., Morganella spp., Proteus spp., Providencia spp.) from different Brazilian hospitals. A total of 4000 carbapenem-resistant Enterobacteriaceae isolates were collected from clinical samples in 50 Brazilian hospitals during 2013-2015. Of these, 499 carbapenem-resistant isolates (CLSI criteria) were selected for further evaluation via broth microdilution to assess for the activity of plazomicin, colistin, tigecycline, meropenem, amikacin, and gentamicin. Additionally, the isolates were assessed for the presence of carbapenemase genes (blaKPC, blaNDM, blaOXA-48-like, blaIMP, blaBKC, blaGES, and blaVIM) by polymerase chain reaction (PCR). When PCR was positive to blaOXA-48-like, blaIMP, blaGES, and blaVIM, the carbapenemase genes were sequenced. blaKPC was the most prevalent carbapenemase gene found (n=397), followed by blaNDM (n=81), blaOXA-48 (n=12), and blaIMP-1 (n=3). Other genes were identified in only 1 isolate each: blaBKC-1, blaGES-16, blaGES-1, blaOXA-370, and blaVIM-1. One isolate had 2 carbapenemase genes (blaKPC and blaNDM). Thirty-three percent of the isolates were nonsusceptible to colistin, 24% to tigecycline, 97% to meropenem, 51% to amikacin, and 81% to gentamicin (via EUCAST criteria). The plazomicin MIC50/90 was 0.5/64mg/L, with 85% of MICs ≤2mg/L and 87% of MICs ≤4mg/L. Elevated MICs to plazomicin were not associated with a specific carbapenemase or bacterial species. The MICs of plazomicin against CPE were lower than those of other aminoglycosides. Plazomicin is a promising drug for the treatment of CPE infections.

Acquisition of the mcr-1 gene by a high-risk clone of KPC-2-producing Klebsiella pneumoniae ST437/CC258, Brazil.

We identified one clinical isolate of K. pneumoniae harboring the mcr 1 (plasmid of IncX4 family) and blaKPC-2 (plasmid of IncFIB family) genes in southern Brazil. These findings highlight that K. pneumoniae isolates carrying both mcr-1 and blaKPC-2 may emergence as a serious threat to antimicrobial therapy.

Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae

Abstract Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to β-lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.

Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae.

Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to ß-lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.

Detection of Staphylococcus aureus with an intermediate profile to vancomycin (VISA) isolate from Santa Maria, RS.

Twenty-three isolates of Staphylococcus aureus resistant to methicillin have been analyzed, being found a clinical isolate of VISA through microdilution technique. The others techniques were unable to detect such isolates. This is the first study that shows the presence of VISA in clinical isolates in the city of Santa Maria-RS.

Chlorhexidine activity against bacterial biofilms.

BACKGROUND: A biofilm is a complex microbiological ecosystem deposited on surfaces. Microorganisms in form of biofilms are of particular clinical concern because of the poor response to antimicrobial treatments. This study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial activity of chlorhexidine, a powerful antiseptic widely used in the hospital environment. METHODS: Disk diffusion and susceptibility tests were conducted in accordance with Clinical and Laboratory Standards Institute standards for the determination of biofilm inhibitory concentration. Chlorhexidine was tested first at a minimum inhibitory concentration and then at higher concentrations when it was not able to destroy the biofilm. The plates were developed with a solution of 0.1% crystal violet, and readings were made at an optical density of 570 nm. RESULTS: Chlorhexidine demonstrated excellent antimicrobial activity for most microorganisms tested in their free form, but was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. CONCLUSION: This study confirms that microorganisms in biofilms have greater resistance to chlorhexidine, likely owing to the mechanisms of resistance conferred to the structure of biofilms.

Identification of mycobacteria isolated at University Hospital of Santa Maria, Rio Grande do Sul, Brazil / Identificação de micobactérias isoladas no Hospital Universitário de Santa Maria, Rio Grande do Sul, Brasil

This study evaluated the prevalence of nontuberculous mycobacterium (NTM) in relation to the total number of cases of mycobacterial infections detected in patients admitted at the University Hospital of Santa Maria from 2008 to 2010. From the positive samples for the genus Mycobacterium, 67% belonged to the Mycobacterium tuberculosis complex (MTBC) and 33% of them were classified as NTM. This investigation aims to contribute to the epidemiology of mycobacterioses, inasmuch as patients infected by NTM require distinctive treatment and monitoring in comparison with those infected by MTBC.

Foi avaliada a prevalência de micobactérias não tuberculosas (MNT) em relação ao total de casos de micobacterioses identificadas em pacientes do Hospital Universitário de Santa Maria, entre os anos de 2008 e 2010. Entre as amostras positivas para o gênero Mycobacterium, 67% eram do complexo Mycobacterium tuberculosis (CMTB) e 33% foram classificadas como MNT. Este estudo procura contribuir com a epidemiologia das micobacterioses, uma vez que os pacientes infectados por MNT necessitam de tratamento e acompanhamento diferenciado dos infectados pelo CMTB.