Hospital sewage in Brazil: a reservoir of multidrug-resistant carbapenemase-producing Enterobacteriaceae.

The One Health concept recognizes that human health is clearly linked to the health of animals and the environment. Infections caused by bacteria resistant to carbapenem antibiotics have become a major challenge in hospitals due to limited therapeutic options and consequent increase in mortality. In this study, we investigated the presence of carbapenem-resistant Enterobacteriaceae in 84 effluent samples (42 from hospital and 42 from non-hospital) from Campo Grande, midwest Brazil. First, sewage samples were inoculated in a selective culture medium. Bacteria with reduced susceptibility to meropenem and ertapenem were then identified and their antimicrobial susceptibility was determined using the Vitek-2 system. The blaKPC genes were detected using PCR and further confirmed by sequencing. Carbapenem-resistant Enterobacteriaceae (CRE) were identified in both hospital (n=32) and non-hospital effluent (n=16), with the most common being Klebsiella pneumoniae and of the Enterobacter cloacae complex species. This is the first study to indicate the presence of the blaKPC-2 gene in carbapenem-resistant Enterobacteriaceae, classified as a critical priority by the WHO, in hospital sewage in this region. The dissemination of carbapenem antibiotic-resistant genes may be associated with clinical pathogens. Under favorable conditions and microbial loads, resistant bacteria and antimicrobial-resistance genes found in hospital sewage can disseminate into the environment, causing health problems. Therefore, sewage treatment regulations should be implemented to minimize the transfer of antimicrobial resistance from hospitals.

Investigation of genotyping and phenotyping characteristics of carbapenem-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae (K. pneumoniae) is a major cause of healthcare-associated infections and plays a prominent role in the widespread antibiotic resistance crisis. Accurate identification of carbapenemases is essential to facilitate effective antibiotic treatment and reduce transmission of K. pneumoniae. This study aimed to detect carbapenemase production in carbapenem-resistant K. pneumoniae strains using phenotypic and genotypic methods. A total of 67 carbapenem-resistant K. pneumoniae strains obtained from various clinical samples were utilized for identification and antimicrobial susceptibility by the Vitek 2 Compact system (Biomerieux, France). Carbapenemase production was determined by using the Polymerase chain reaction, Blue-carba test (BCT) and Carbapenem inactivation method (CIM). Out of the isolates, 59 (88.1%) were positive bla OXA-48, 16 (23.9%) bla IMP, and five (7.5%) were positive bla NDM. No bla KPC genes were detected. The CIM identified 62 (92.5%), BCT identified 63 (94%) of PCR-positive isolates. The sensitivity and specificity of the BCT and the CIM were determined to be 96.7%, 40%, and 96.7%, 25% respectively. The bla OXA-48 gene was found to be the most prevalent in K. pneumoniae isolates. Early identification of carbapenem resistance plays a vital role in designing effective infection control strategies and mitigating the emergence and transmission of carbapenem resistance, thus reducing healthcare-associated infections.

Susceptibility evaluation of novel beta-lactam/beta-lactamase inhibitor combinations against carbapenem-resistant Klebsiella pneumoniae from bloodstream infections in hospitalized patients in Brazil.

INTRODUCTION: Novel beta-lactam/beta-lactamase inhibitor (BIBLI) combinations are commercially available and have been used for treating carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Continuous surveillance of susceptibility profiles and resistance mechanism identification are necessary to monitor the evolution of resistance within these agents. OBJECTIVE: The purpose of this study was to evaluate the susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in CRKP isolated from patients with bloodstream infections who underwent screening for a randomized clinical trial in Brazil. METHODS: Minimum inhibitory concentrations (MICs) were determined for meropenem, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam using the gradient diffusion strip method. Carbapenemase genes were detected by multiplex real-time polymerase chain reaction. Klebsiella pneumoniae carbapenemase (KPC)-producing isolates showing resistance to any BLBLI and New Delhi Metallo-beta-lactamase (NDM)-producing isolates with susceptibility to any BLBLI isolates were further submitted for whole-genome sequencing. RESULTS: From a total of 69 CRKP isolates, 39 were positive for blaKPC, 19 for blaNDM and 11 for blaKPC and blaNDM. KPC-producing isolates demonstrated susceptibility rates above 94 % for all BLBLIs. Two isolates with resistance to meropenem/vaborbactam demonstrated a Gly and Asp duplication at the porin OmpK36 as well as a truncated OmpK35. All NDM-producing isolates, including KPC and NDM coproducers, demonstrated susceptibility rates to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam of 0 %, 9.1-21.1 % and 9.1-26.3 %, respectively. Five NDM-producing isolates that presented susceptibility to BLBLIs also had porin alterations CONCLUSIONS: This study showed that, although high susceptibility rates to BLBLIs were found, KPC-2 isolates were able to demonstrate resistance probably as a result of porin mutations. Additionally, NDM-1 isolates showed susceptibility to BLBLIs in vitro.

High-risk clones of carbapenem resistant Klebsiella pneumoniae recovered from pediatric patients in Southern Brazil.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) exhibit high mortality rates in pediatric patients and usually belong to international high-risk clones. This study aimed to investigate the molecular epidemiology and carbapenem resistance mechanisms of K. pneumoniae isolates recovered from pediatric patients, and correlate them with phenotypical data. Twenty-five CRKP isolates were identified, and antimicrobial susceptibility was assessed using broth microdilution. Carbapenemase production and ß-lactamase genes were detected by phenotypic and genotypic tests. Multilocus sequence typing was performed to differentiate the strains and whole-genome sequencing was assessed to characterize a new sequence type. Admission to the intensive care unit and the use of catheters were significantly positive correlates of CRKP infection, and the mortality rate was 36%. Almost all isolates showed multidrug-resistant phenotype, and most frequent resistant gene was blaKPC. We observed the dissemination of ST307 and clones belonging to CG258, which are considered high risk. In pediatric patients, these clones present with high genomic plasticity, favoring adaptation of the KPC and NDM enzymes to healthcare environments.

Detection of Carbapenem Resistance in Enterobacterales Directly From Positive Blood Cultures Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.

CONTEXT.­: Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carbapenemase production, by evaluating carbapenem hydrolysis. OBJECTIVE.­: To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. DESIGN.­: We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene (blaKPC, blaGES, blaNDM-1, blaIMP, blaVIM, and blaOXA-48-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2-4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. RESULTS.­: Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For blaKPC-positive isolates, sensitivity was 97.9%, while it reduced significantly for blaNDM-1 and blaOXA-48-like isolates: 62.5% (10 of 16) and 66.7% (6 of 9), respectively. Indeed, logRQ was higher in blaKPC-positive isolates (0.37-1.97) than in blaNDM-1 (-1.37 to 0.83) and blaOXA-48-like isolates (-1.08 to 1.79). CONCLUSIONS.­: This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures.

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

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

The Presence of Virulent and Multidrug-Resistant Clones of Carbapenem-Resistant Klebsiella pneumoniae in Southeastern Brazil.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) represents an urgent threat to global public health due to the limited therapeutic options available to control this pathogen. This study aims to analyze the molecular epidemiology, antimicrobial resistance and virulence profile of CRKP isolated from patients at hospitals in Southeastern Brazil. KPC and other beta-lactamase genes were detected in all strains, which were also multidrug-resistant (MDR). In addition, 11 strains showed resistance to last-resort antimicrobials, such as colistin and tigecycline. MLST analysis revealed eight different sequence types (ST11, ST37, ST147, ST340, ST384, ST394, ST437, and ST628), being two (ST628 and ST394) reported for the first time in Brazil. Strains belonging to the clonal complex 258 (CC258) "high-risk clones" were prevalent in this study. The Galleria mellonella model showed the emergence of virulent CRKP strains in the healthcare environment and, suggests that colistin-resistant strains were associated with higher virulence. This study shows the presence of virulent CRKP-MDR strains in hospitals across Southeastern Brazil, and draws attention to the presence of highly virulent emerging CRKP-MDR ST628 strains, showing that virulent and resistant clones can emerge quickly, requiring constant monitoring.

Performance of two methods of carbapenem-resistant Enterobacterales surveillance on a kidney transplant ward: selective culture of and real-time PCR directly from rectal swabs.

BACKGROUND: Infection with carbapenem-resistant Enterobacterales (CRE) is associated with a high mortality rate in kidney transplant recipients, and colonization with CRE is one of the major risk factors for CRE infection. There is, therefore, a need to improve the capacity to detect colonization with CRE among inpatients. METHODS: In this prospective study, we compared the performance of real-time PCR for carbapenemase directly from rectal swabs with that of conventional CRE surveillance culture in all patients admitted to a kidney transplant ward between February 2019 and March 2020. Surveillance culture and real-time PCR were performed at admission and weekly until hospital discharge. Two perineum-rectal swabs were collected: one for culture and one for PCR. RESULTS: We collected 905 paired samples for CRE surveillance from 399 patients, of whom 347 (87.0%) were kidney transplant recipients and 52 were waiting list patients. CRE was detected by culture and/or PCR in 75 patients (18.8%). Positivity for CRE was identified by PCR in 62 (15.5%) of the 399 patients and by culture in 55 (13.8%); 20 (5.0%) of the patients tested positive only on PCR, and 13 (3.3%) tested positive only on culture. The most common carbapenemase and species were, respectively, blaKPC (in 85.5%) and Klebsiella pneumoniae (in 80.0%). Infection with CRE occurred in 21.6% of the colonized patients, those cases occurred only among kidney transplant recipients. None of the patients who tested negative on culture developed CRE infection. CONCLUSION: In conclusion, the two methods are complementary and could be useful in a scenario of high CRE prevalence.

High mortality from carbapenem-resistant Klebsiella pneumoniae bloodstream infection.

In this study, it was evaluated clinical data of 107 patients with bloodstream infection (BSI) by Klebsiella pneumoniae and performed phenotypic and molecular analyzes in 50.5% (54/107) of the samples, those that showed a resistance profile to carbapenemics. The blaKPC gene was present in 90.4% (49/54) of the samples, blaNDM gene in one sample and, in 7.4% (4/54) of the samples, no carbapenemase gene was found. In the similarity analysis, it was found 4 main clones and 11 samples were not genetically related. The median age of the patients was 58 (40-70) years old and 60.7% (65/107) were male. When comparing two groups of patients with BSI due to K. pneumoniae with and without resistance to carbapenems, the variables ICU permanence, renal failure (IR), previous use of antimicrobials, Charlson's comorbidity index (ICCi), some invasive procedures and death showed a statistically significant difference (p < 0.05). And when relating death as a dependent variable, IR, liver failure and patients with BSI XDR or PDR, were predictors of increased mortality. Our study showed a higher mortality rate in patients with BSI due to carbapenem-resistant pneumonia with additional resistance or not to polymyxins.

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

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

Draft genome sequences of two hypermucoviscous carbapenem-resistant ST25 Klebsiella pneumoniae strains causing respiratory and systemic infections.

OBJECTIVES: The emergence and spread of hypermucoviscous KPC-2-producing Klebsiella pneumoniae strains belonging to the sequence type 25 (ST25) clone was reported recently in Northwest Argentina as a leading cause of nosocomial infections. The aim of this work was to perform whole-genome sequencing (WGS) to analyse antimicrobial resistance genes (ARGs), virulence factors and colonisation-associated genes in two carbapenem-resistant KPC-2-producing ST25 K. pneumoniae strains isolated from hospitalised patients. METHODS: Classical microbiological methods were applied to recover K. pneumoniae LABACER 01 from a bone sample and LABACER 27 from the respiratory tract of two hospitalised patients. Bacteria were identified by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). WGS was performed using an Illumina MiSeq platform. Genome annotation and analysis were performed with available databases and bioinformatic tools. RESULTS: Genomic analysis revealed a genome of 5 598 020 bp with 19 further characterised ARGs in strain LABACER 01, and a genome of 5 622 382 bp with 20 ARGs in strain LABACER 27. Bioinformatics analysis also predicted genomic regions associated with virulence factors and mucosal tissue colonisation. CONCLUSION: This study reports the genomic analysis of K. pneumoniae LABACER 01 and LABACER 27, two hypermucoviscous carbapenem-resistant ST25 strains, which expands our knowledge on the antibiotic resistance, pathogenic mechanisms and biology of ST25 clones recently emerging in Argentina.

A multispecies outbreak of carbapenem-resistant bacteria harboring the blaKPC gene in a non-classical transposon element.

BACKGROUND: Klebsiella pneumoniae is the most frequent KPC-producing bacteria. The blaKPC gene is frequently embedded in Tn4401 transposon, and less frequently in non-Tn4401 elements (NTEKPC) variants I-III. The first case of KPC in the UC-CHRISTUS Clinical Hospital was detected in Pseudomonas aeruginosa. Soon after this event, KPC was detected in 2 additional Pseudomonas aeruginosa, 3 Escherichia coli, 3 Enterobacter cloacae, 3 Klebsiella pneumoniae, and 1 Citrobacter freundii, isolated from 6 different patients. We aimed to elucidate the possible mechanisms of genetic transfer and dissemination of the blaKPC gene among isolates of this multispecies outbreak. A molecular epidemiology analysis of the above mentioned clinical isolates (n = 13) through Multi-Locus Sequence Typing, plasmid analysis, Pulsed-Field Gel-Electrophoresis, and Whole-genome sequencing (WGS) was performed. RESULTS: High-risk sequence types were found: K. pneumoniae ST11, P. aeruginosa ST654, and E. cloacae ST114. All enterobacterial isolates were not clonal except for 3 E. coli isolated from the same patient. WGS analysis in 6 enterobacterial isolates showed that 4 of them had blaKPC embedded in a novel variant of NTEKPC designated NTEKPC-IIe. Upstream of blaKPC gene there was a 570 pb truncated blaTEM-1 gene followed by an insertion sequence that was 84% similar to ISEc63, a 4473 bp element of the Tn3 family. Downstream the blaKPC gene there was a truncated ISKpn6 gene, and the inverted repeat right sequence of Tn4401. The ISec63-like element together with the blaKPC gene plus Tn4401 remnants were inserted in the Tra operon involved in conjugative transfer of the plasmid. This NTE was carried in a broad host-range IncN plasmid. P. aeruginosa isolates carried blaKPC gene embedded in a typical Tn4401b transposon in a different plasmid, suggesting that there was no plasmid transfer between Enterobacteriaceae and P. aeruginosa as initially hypothesized. CONCLUSIONS: Most enterobacterial isolates had blaKPC embedded in the same NTEKPC-IIe element, suggesting that this multispecies KPC outbreak was due to horizontal gene transfer rather than clonal spread. This poses a greater challenge to infection control measures often directed against containment of clonal spread.

Molecular investigation of an outbreak associated with total parenteral nutrition contaminated with NDM-producing Leclercia adecarboxylata.

BACKGROUND: This study aimed to determine the epidemiological, microbiological, and molecular characteristics of an outbreak of carbapenem-resistant Leclercia adecarboxylata in three hospitals associated with the unintended use of contaminated total parental nutrition (TPN). METHODS: For 10 days, 25 patients who received intravenous TPN from the same batch of a formula developed sepsis and had blood cultures positive for L. adecarboxylata. Antimicrobial susceptibility and carbapenemase production were performed in 31 isolates, including one from an unopened bottle of TPN. Carbapenemase-encoding genes, extended-spectrum ß-lactamase-encoding genes were screened by PCR, and plasmid profiles were determined. Horizontal transfer of carbapenem resistance was performed by solid mating. Clonal diversity was performed by pulsed-field gel electrophoresis. The resistome was explored by whole-genome sequencing on two selected strains, and comparative genomics was performed using Roary. RESULTS: All 31 isolates were resistant to aztreonam, cephalosporins, carbapenems, trimethoprim/sulfamethoxazole, and susceptible to gentamicin, tetracycline, and colistin. Lower susceptibility to levofloxacin (51.6%) and ciprofloxacin (22.6%) was observed. All the isolates were carbapenemase producers and positive for blaNDM-1, blaTEM-1B, and blaSHV-12 genes. One main lineage was detected (clone A, 83.9%; A1, 12.9%; A2, 3.2%). The blaNDM-1 gene is embedded in a Tn125-like element. Genome analysis showed genes encoding resistance for aminoglycosides, quinolones, trimethoprim, colistin, phenicols, and sulphonamides and the presence of IncFII (Yp), IncHI2, and IncHI2A incompatibility groups. Comparative genomics showed a major phylogenetic relationship among L. adecarboxylata I1 and USDA-ARS-USMARC-60222 genomes, followed by our two selected strains. CONCLUSION: We present epidemiological, microbiological, and molecular evidence of an outbreak of carbapenem-resistant L. adecarboxylata in three hospitals in western Mexico associated with the use of contaminated TPN.

Molecular characterization of KPC-2-producing Klebsiella pneumoniae ST258 isolated from bovine mastitis.

Bovine mastitis, an inflammation of the mammary gland of dairy cattle, is the most prevalent disease causing economically important losses, reduced milk production, early culling, veterinary expenses, and higher death rates. Bovine mastitis infections are the main cause for the use of antibiotics; however, the emergence of multidrug-resistant bacteria and the poor or nil response to antibiotics has become a critical global health problem. The goal of this study was the characterization of bacterial infections associated with clinical bovine mastitis. All the isolates were multidrug-resistant and were negative for the production of extended spectrum ß-lactamases. However, all isolates were identified as carbapenemase-producing organisms by the Carba NP test. The carbapenemase identified was the product of the KPC-2 gene. The isolates were identified as Klebsiella pneumoniae and contained virulence genes for fimbriae, lipopolysaccharides, nitrogen starvation genes, and siderophores. Sixty-nine percent of the KPC-2-producing isolates had the same plasmid profile, although the genetic mobilization of resistance by bacterial conjugation was unsuccessful. The carbapenemase corresponded to the plasmid-borne KPC-2 gene identified by Southern blot hybridization. The assay showed a positive signal in the 90 kb (69% of the isolates), 165 kb (31% of the isolates), and 130 kb (6% of the isolates) plasmids. The IncFIIy and IncFIIk replicons were detected among these K. pneumoniae isolates. The PFGE and MLST analysis showed that all of the isolates are comprised by two clones (A and B) belonging to Sequence Type 258. This is the first report of K. pneumoniae producing carbapenemase KPC-2 isolated from bovine mastitis.

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.

In Vivo High Plasticity of Multi-Drug Resistant ST258 Klebsiella pneumoniae.

Carbapenemase production in Enterobacterales clinical isolates is a global threat. Multi-drug resistant Klebsiella pneumoniae harboring carbapenemases are a major concern among the hospital settings in Latin America. Aim: The aim of this study was to analyze the genetic relatedness between three isolates of K. pneumoniae recovered from one patient in the same bacteriological round on the same day, which exhibited different susceptibility profiles to carbapenems (CP) and to colistin (Col). Isolates' profiles were as follows (susceptible-S/resistant-R): CPS/ColR, CPR/ColR, and CPR/ColS. Pulse-field gel electrophoresis, multilocus sequence typing, and whole genome sequencing were performed. Conjugation assays were carried out and PCR determination in transconjugants (Tcs) was made for: blaCTX-M-groups, blaNDM, blaKPC, blaTEM, qnr alleles, aac(6')Ib-cr, ermB, and plasmid incompatibility groups (Inc). Results: All isolates belonged to the same clone, to ST258 and harbored blaCTX-M-14, blaCTX-M-15, qnrA1, qnrB1, aac(6')Ib-cr, and wzi154 (capsule-locus KL107). One isolate had additional wzi gene, wzi109 (capsule-locus KL36). In CPR isolates, the pattern was explained for blaNDM-1 or blaNDM-1/blaKPC-2 presence, and in ColR for IS5-like element insertion in mgrB at different positions. Co-mobilization of blaNDM-1/qnrA1 was associated to a different plasmid Inc (A/C-FII) in both blaNDM-1 donors. Mobilization of blaCTX-M-14 was related to IncI1 in one donor. Conclusion: These findings highlight the potential plasticity of ST258 K. pneumoniae clone. To the best of our knowledge, this is the first description of blaNDM-1/blaKPC-2-producing K. pneumoniae ST258 in Latin America.

The influence of hospital antimicrobial use on carbapenem-non-susceptible Enterobacterales incidence rates according to their mechanism of resistance: a time-series analysis.

BACKGROUND: Carbapenem non-susceptible Enterobacterales (CNSE) can be broadly divided into those that produce carbapenemases (carbapenemase-producing Enterobacterales (CPE)), and those that harbour other mechanisms of resistance (non-carbapenemase-producing CNSE (NCP-CNSE)). AIM: To determine the predictors of CNSE nosocomial incidence rates according to their mechanism of resistance. METHODS: A time-series analysis was conducted (July 2013 to December 2018) to evaluate the relationship in time between hospital antibiotic use and the percentage of adherence to hand hygiene with the CNSE rates. FINDINGS: In all, 20,641 non-duplicated Enterobacterales isolates were identified; 2.2% were CNSE. Of these, 48.1% and 51.9% were CPE and NCP-CNSE, respectively. Of the CPE, 78.3% possessed a blaOXA-232 gene. A transfer function model was identified for CNSE, CPE, and OXA-232 CPE that explained 20.8%, 19.3%, and 24.2% of their variation, respectively. According to the CNSE and CPE models, an increase in piperacillin-tazobactam (TZP) use of 1 defined daily dose (DDD) per 100 hospital patient-days (HPD) would lead to an increase of 0.69 and 0.49 CNSE and CPE cases per 10,000 HPD, respectively. The OXA-232 CPE model estimates that an increase of 1 DDD per 100 HPD of TZP use would lead to an increase of 0.43 OXA-232 CPE cases per 10,000 HPD. A transfer function model was not identified for NCP-CNSE, nor was there an association between the adherence to handhygiene and the CNSE rates. CONCLUSION: The use of TZP is related in time with the CPE nosocomial rates, mostly explained by its effect on OXA-232 CPE.

Genetic Relatedness of NDM-Producing Klebsiella pneumoniae Co-Occurring VIM, KPC, and OXA-48 Enzymes from Surveillance Cultures from an Intensive Care Unit.

Klebsiella pneumoniae is an opportunistic bacterial pathogen most commonly associated with nosocomial infections, especially in intensive care unit (ICU) patients. Routine surveillance cultures for carbapenemase-producing Enterobacteriaceae have become a common practice for hospital infection prevention. The objective of this study was to investigate the genetic relatedness of carbapenem-resistant K. pneumoniae carrying blaNDM gene across multilocus sequence typing (MLST) scheme. Surveillance rectal swabs from 4,463 ICU patients admitted to the Rio de Janeiro hospital (March 2016-2017) were screened on CHROMagar mSuperCARBA. Of these, 631 isolates were subjected to VITEK 2 system for phenotypic microbial identification and antibiotic susceptibility testing. Out of 631 isolates, 108 were identified as K. pneumoniae, 103 of which were confirmed by PCR of 16S-23S rDNA internal transcribed spacer (ITS). Eleven blaNDM-positive isolates were subsequently screened for blaKPC, blaBKC, blaIMP, blaVIM, blaSPM, blaOXA-48, and mcr-1-8 genes. Twenty-seven percent (3/11) revealed co-occurrence with KPC, OXA-48, and VIM, 46% (5/11) with KPC and VIM, and 18% (2/11) with VIM type. No strains harbored the blaBKC, blaSPM, blaIMP, and mcr-1 to 8 resistance genes. All 11 isolates were resistant to ß-lactams, ciprofloxacin 90%, tigecycline 82%, gentamicin 73%, and amikacin 18%, and were classified as multidrug resistant (MDR), extensively drug resistant (XDR), and pandrug resistant (PDR). Molecular epidemiology data based on MLST revealed 11 different STs, 8 of which were novel and 3 were previously described. Six out of the eight new STs were associated with MDR and PDR strains and two clonal complexes were reported, including CC258 and CC15. The coexistence of NDM-producing K. pneumoniae and other carbapenemase has been frequently described worldwide. Moreover, we report for the first time K. pneumoniae co-harboring up to four carbapenemases from active surveillance cultures.

Diagnostic performance of the Xpert Carba-R™ assay directly from rectal swabs for active surveillance of carbapenemase-producing organisms in the largest Brazilian University Hospital.

BACKGROUND: The global spread of carbapenemase-producing organisms (CPO) has been considered by international health authorities as a critical public health concern. Brazil has a high CPO prevalence according to distinct publications but many routine microbiology laboratories have only phenotypic resources to evaluate this epidemiological situation, which is time-consuming and detects only carbapenem-resistant isolates missing CPO susceptible expressing a slightly decreased susceptibility. New molecular platforms can detect CPO faster but a local evaluation is essential. AIM: To evaluate the performance of CPO detection direct from rectal swabs with the Xpert Carba-R™ assay (Cepheid, Sunnyvale, CA) in the largest Brazilian University Hospital. METHODS: A prospective diagnostic accuracy study of CPO was performed with the collection of rectal swabs from patients admitted into the Intensive Care Unit (ICU) and into the Emergency Department (ED) between April and July 2016. The Xpert Carba-R™ assay results were compared with carbapenem-resistant Enterobacterales (CRE) surveillance cultures plus in-house PCR carbapenemase detection (reference method). In case of discordant results between methods, additional tests were performed. The limit of detection (LoD) for the CRE culture and the Xpert Carba-R™ assay were performed with contrived isolates of known carbapenemases genes. RESULTS: A total of 921 clinical rectal swabs were analyzed being 21% (196/921) from the ICU and 79% (725/921) from the ED. Overall, the Xpert Carba-R™ assay detected 9.9% (91/921) of CPOs being 9.5% (87/921) positive only for blaKPC and 0.4% (4/921) positive only for blaNDM. The reference method detected 9.1% (84/921) CPO being 77 (8.4%) blaKPC, 5 blaVIM (0.5%) and 2 blaNDM (0.2%). No IMP or OXA-48 like gene was detected. Overall, twelve samples, 1.3% (10 blaKPC, 2 blaNDM) were Xpert Carba-R™ positive but negative by the reference method. Five isolates (0.5%) were positive for blaVIM only by in-house PCR and confirmed to be blaVIM-2 by DNA sequencing. The Kappa value, sensitivity, specificity, positive/negative predictive values and accuracy of the Xpert Carba-R™ assay were; 0.893 (95% confidence interval [CI], 0.842-0.944), 94% (86.7-98.0), 98.6% (97.5-99.3), 86.8% (78.1-93.0), 99.4% (98.6-99.8) and 98.2% (97.3-99.1), respectively. The LoD for blaKPC of the Xpert Carba-R™ assay and the CRE cultures were 101 CFU/swab. CONCLUSION: The Xpert Carba-R™ assay is an accurate test to detect CPO directly from the rectal swabs with significant lower turnaround time (TAT) when compared to the reference method (CRE culture plus in-house PCR). Xpert Carba-R™ may, therefore, be regarded as a good and fast epidemiological tool.

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.