Detection of KPC enzyme by MALDI-TOF MS from bacteria impregnated in filter paper.

The main mechanism that causes resistance to carbapenem, one of the most potent antibiotic available, in Enterobacterales bacterial isolates, is due to Klebsiella pneumoniae carbapenemase (KPC) production by the bacterium. KPC is spread worldwide, requiring laboratories to be capable of identifying this enzyme, however some methods can be expensive for small laboratories, especially in developing countries. Therefore, the development of methods with low cost of reagents for the detection of KPC enzyme is necessary. The objective of this study was to evaluate the detection of KPC enzyme by MALDI-TOF MS from inactivated bacteria impregnated in filter paper. A total of 129 Enterobacterales isolates were impregnated in filter paper, and after 7 days at room temperature, they were subjected to a protein extraction protocol and spectra acquisition, in triplicates, by MALDI-TOF MS. The spectra were evaluated and KPC was identified according to the presence of a peak of 28,712.62 ± 27.80 m/z. Considering the presence of the KPC peak in at least one spectrum of the triplicates, this method presented 60.8% sensitivity and 96.4% specificity. However, considering the presence of KPC peak in at least two spectra of the triplicate, a specificity of 100% was achieved. The detection of KPC enzyme from inactivated bacteria impregnated in filter paper can be used as a method to confirm the presence of KPC, which could be very significant for small laboratories with limited resources.

Escherichia coli carrying blaNDM-1 obtained from a migratory penguin (Spheniscus magellanicus) in the Brazilian seacoast.

The reservoirs for NDM-producing Enterobacterales are increasing, not only in hospitals, but also in the environment and in the community, challenging the therapeutic efficacy of carbapenems. We aimed to characterize an isolate of Escherichia coli harboring the blaNDM-1 gene recovered from the bloodstream of a penguin (Spheniscus magellanicus) in Southern Brazil. A total of 74 bacterial isolates recovered from arterial blood samples from dead birds were submitted to species identification and antibiotic susceptibility evaluation. One isolate presented resistance to carbapenems (E. coli 89PenNDM) and proved to harbor the blaNDM-1 gene by multiplex high-resolution melting real-time PCR (PCR-HRM). Conjugation experiments indicated that the blaNDM-1 was transmissible to E. coli J53. Whole genome sequencing (WGS) confirmed the presence of the blaNDM-1 gene in a conjugative plasmid (IncA/C2 plasmid) in both the E. coli 89PenNDM and its transconjugants. The isolate was classified as ST 156 and many other resistance genes (e.g., sul1, sul,2, strA, floR, tet(A)) were identified, all carried in the same IncA/C2 plasmid. This is the first report of blaNDM-1-producing E. coli isolated from a penguin in the Brazilian seacoast. The presence of a carbapenemase gene in wildlife animals is of concern as they may become reservoirs of multidrug-resistant bacteria and disseminate them to the environment.

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.

Polymyxin B broth disk elution: a feasible and accurate methodology to determine polymyxin B susceptibility in Enterobacterales.

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 µg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 µg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales.

Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five ß-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae.

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While ß-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a blaKPC-2 and several other ß-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular ß-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our ß-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of ß-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While ß-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of ß-lactams leads to high drug concentrations at their periplasmic target sites, allowing ß-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of ß-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five ß-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem ß-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of ß-lactams and combat carbapenem-resistant isolates.

High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS.

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

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.

Dissemination of blaOXA-370 is mediated by IncX plasmids and the Tn6435 transposon.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

KPC-producing Klebsiella pneumoniae bloodstream isolates from Brazilian hospitals: What (still) remains active?

OBJECTIVES: This study assessed susceptibility to polymyxin B (PMB) and alternative antimicrobials, with focus on aminoglycosides and tigecycline, according to different breakpoints in KPC-producing Klebsiella pneumoniae (KPC-Kp) bloodstream isolates from Brazilian hospitals. METHODS: Bloodstream K. pneumoniae isolates non-susceptible to any of the three carbapenems (meropenem, imipenem or ertapenem) from four Brazilian tertiary-care hospitals were selected. Antimicrobial susceptibility was determined and interpreted according to distinct breakpoints. Twenty-nine PMB-resistant KPC-Kp isolates were selected for molecular typing. RESULTS: A total of 158 KPC-Kp were analysed. MIC50/90 values for PMB were 0.25/16mg/L; 40 isolates (25.3%) were resistant to PMB. MIC50/90 values for meropenem were 32/≥256mg/L; no isolates were susceptible to meropenem according to CLSI, but 10 isolates were intermediate using EUCAST breakpoints (1, MIC=4mg/L; 9, MIC=8mg/L). MIC50/90 values for tigecycline were 2/8mg/L; 53 (33.5%) and 94 (59.5%) isolates were susceptible according to EUCAST and FDA breakpoints, respectively. MIC50/90 values were 32/≥64mg/L for amikacin and ≥16/≥16mg/L for gentamicin; 48 (30.4%), 28 (17.7%) and 16 (10.1%) were susceptible to amikacin according to CLSI, EUCAST and USCAST, respectively, but susceptibility rates to gentamicin were <7.0%. Eighteen distinct clonal profiles were identified among 29 PMB-resistant isolates by DNA macrorestriction. Most clones belonged to CC11. CONCLUSION: Elevated rates of PMB-resistant KPC-Kp bloodstream infections were found in four Brazilian hospitals, mostly of polyclonal origin. Alternative antimicrobials with the highest in vitro activity were tigecycline and amikacin, although susceptibility rates significantly decreased using criteria with stricter breakpoints (e.g. EUCAST, USCAST).

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.

In vitro antimicrobial activity of imipenem plus amikacin or polymyxin B against carbapenem-resistant Pseudomonas aeruginosa isolates.

The aim of this study was to evaluate the antimicrobial activity of imipenem plus amikacin or polymyxin B against six carbapenem-resistant P. aeruginosa by time-kill assay. Synergism was observed in two isolates for combinations with amikacin. For combinations with polymyxin B, synergism and antagonism also occurred in two isolates.

PCR Assay Based on the gyrB Gene for Rapid Identification of Acinetobacter baumannii-calcoaceticus Complex at Specie Level.

BACKGROUND: The genus Acinetobacter sp. comprises more than 50 species, and four are closely related and difficult to be distinguished by either phenotypic or genotypic methods: the Acinetobacter calcoaceticus-baumannii complex (ABC). The correct identification at species level is necessary mainly due to the epidemiological aspects. METHODS: We evaluated a multiplex PCR for gyrB gene to identify the species of the ABC using the sequencing of the ITS 16S-23S fragment as a gold standard. Isolates identified as Acinetobacter calcoaceticus-baumannii from three hospitals at southern Brazil in 2011 were included in this study. RESULTS: A total of 117 isolates were obtained and 106 (90.6%) were confirmed as A. baumannii, 6 (5.1%) as A. nosocomialis and 4 (3.4%) as A. pittii by PCR for gyrB gene. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS. We also noted that the non-A. baumannii isolates were recovered from respiratory tract (8/72.7%), blood (2/18.2%) and urine (1/9.1%), suggesting that these species can cause serious infection. CONCLUSION: These findings evidenced that the multiplex PCR of the gyrB is a feasible and simple method to identify isolates of the ABC at the species level.

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil.

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.

The changing epidemiology of Acinetobacter spp. producing OXA carbapenemases causing bloodstream infections in Brazil: a BrasNet report.

We evaluated the epidemiology of Acinetobacter spp. recovered from patients diagnosed with bloodstream infections in 9 tertiary hospitals located in all Brazilian geographic regions between April and August 2014. Although OXA-23-producing Acinetobacter baumannii clones were disseminated in most hospitals, it was observed for the first time the spread of OXA-72 among clonally related A. baumannii isolated from distinct hospitals. Interestingly, Acinetobacter pittii was the most frequent species found in a Northern region hospital. Contrasting with the multisusceptible profile displayed by A. pittii isolates, the tetracyclines and polymyxins were the only antimicrobials active against all A. baumannii isolates.

Outbreaks due to Mycobacterium abscessus subsp. bolletii in southern Brazil: persistence of a single clone from 2007 to 2011.

Outbreaks associated with rapidly growing mycobacteria (RGM) have been increasingly reported worldwide, including in Brazil. Among the RGM, the Mycobacterium abscessus complex is the most pathogenic and related to multidrug resistance. The aim of this study was to evaluate the antimicrobial susceptibility and molecular profile of RGM isolates involved in new postsurgical infection outbreaks in Brazil since 2007. Of the 109 cases reported in the state of Rio Grande do Sul between 2007 and 2011, 43 (39 %) had confirmed mycobacterial growth in culture. Clinical isolates were obtained from biopsy specimens or abscess aspirates. PRA-hsp65 restriction pattern identified the isolates as M. abscessus type 2, and partial rpoB sequencing confirmed the identification as M. abscessus subsp. bolletii. All isolates were susceptible to amikacin and resistant to ciprofloxacin, doxycycline, sulfamethoxazole, moxifloxacin and tobramycin. Most isolates (72 %) were fully susceptible to cefoxitin but six isolates (14 %) were fully resistant to clarithromycin. The latter differed from the susceptibility profiles of the previously described BRA100 clone from other Brazilian regions. Nevertheless, pulsed-field gel electrophoresis analysis revealed that these isolates belonged to a single BRA100 clone. In conclusion, our study reports the persistence of an emergent single and highly resistant clone of M. abscessus subsp. bolletii for several years even after national implementation of infection control measures.