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Carbapenem-resistant Enterobacteriaceae are a worldwide health problem and isolates carrying both bla KPC-2 and bla NDM-1 are unusual. Here we describe the microbiological and clinical characteristics of five cases of bloodstream infections (BSI) caused by carbapenem-resistant Klebsiella pneumoniae and Serratia marcescens having both bla KPC-2 and bla NDM-1 . Of the five blood samples, three are from hematopoietic stem cell transplantation patients, one from a renal transplant patient, and one from a surgical patient. All patients lived in low-income neighbourhoods and had no travel history. Despite antibiotic treatment, four out of five patients died. The phenotypic susceptibility assays showed that meropenem with the addition of either EDTA, phenylboronic acid (PBA), or both, increased the zone of inhibition in comparison to meropenem alone. Molecular tests showed the presence of bla KPC-2 and bla NDM-1 genes. K. pneumoniae isolates were assigned to ST258 or ST340 by whole genome sequencing. This case-series showed a high mortality among patients with BSI caused by Enterobacteriae harbouring both carbapenemases. The detection of carbapenemase-producing isolates carrying both bla KPC-2 and bla NDM-1 remains a challenge when using only phenotypic assays. Microbiology laboratories must be alert for K. pneumoniae isolates producing both KPC-2 and NDM-1.

Background: Although guidelines recommend the use of chlorhexidine gluconate (CHG) for hand hygiene (HH), the impact of its routine use on antimicrobial resistance is not clear. Objective: To analyze the impact on the CHG susceptibility among isolates obtained from hands of HCW during its routine use for HH. Methods: We conducted a crossover study at 4 medical-surgical wards of a tertiary-care hospital in São Paulo, Brazil. In 2 units (intervention group), we established routine use of CHG for HH. For the other 2 units (control group), regular soap was provided. The availability of alcohol formulation for HH was not changed during the study. Every 4 months we swapped the units, ie, those using CHG changed for regular soap and vice versa. At baseline, we cultured the hands of HCWs. Only nursing staff hands were investigated. For hand culturing, HCWs placed their hands inside a sterile bag containing a solution of phosphate-buffered saline, Tween 80, and sodium thiosulfate. After the solution incubated overnight, it was inoculated onto brain-heart infusion. Next, it was plated on McConkey and Mannitol agar. MALDI-TOF was used for identification. Agar dilution was performed for Staphylococcus spp. We selected all Staphylococcus spp with MIC ≥ 8 and performed inhibition of efflux pump test. For isolates that showed a decrease of 2 dilutions, we searched the gene qac A/B by polymerase chain reaction. Results: We obtained 262 samples from HCW hands yielding 428 isolates. The most frequent genera were Staphylococcus spp (58%), Acinetobacter spp (8%), Enterobacter spp (8%), Stenotrophomonas spp (5%), Klebsiella spp (4%), Pseudomonas spp (3%), and others (14%). Staphylococcus spp were less frequent in the intervention compared to control group (43% vs 61%; OR, 0.48; 95% CI, 0.29–0.69; P = .005). Among all Staphylococcus spp, the proportion of chlorhexidine resistance (RCHG; MIC ≥ 8) was 12%. All resistant isolates recovered susceptibility after inoculation with pump-efflux inhibitor. For pump-inhibited isolates, 53% had the gene qac A/B amplified by PCR. We did not investigate RCHG among gram-negative isolates. There was a nonsignificant increase in Staphylococcus spp RCHG in the intervention group (4% to 6%; P = .90). Healthcare-acquired infection rates did not change significantly during the intervention. The consumption of CHG increased from 7.3 to 13.9 mL per patient day. Conclusions: We did not detect a significant difference in RCHG during the routine use of CHG for HH, although we observed increasing resistance. Further investigation is needed to clarify other reasons for increasing MIC to CHG. Funding: None Disclosures: None

Background Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Methods Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. Results A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored bla KPC, bla TEM and /or bla CTX associated with decreased or lost of 35–36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae . Conclusions There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen in the last decade. Increased resistance to sulfamethoxazole/trimethoprim (SMX/TMP) has been reported in S. maltophilia strains in the past few years, leading to few therapeutic options. We conducted a prospective multicenter study at two Brazilian teaching hospitals that identified S. maltophilia isolates and evaluated their antimicrobial susceptibility profile, SMX/TMP resistance genes and their clonality profile. A total of 106 non-repeated clinical samples of S. maltophilia were evaluated. Resistance to SMX/TMP was identified in 21.6% of the samples, and previous use of SMX/TMP occurred in 19 (82.6%). PCR detected the sul1 gene in 14 of 106 strains (13.2%). Of these isolates, nine displayed resistance to SMX/TMP. The resistant strains presented a polyclonal profile. This opportunistic pathogen has emerged in immunocompromised hosts, with few therapeutic options, which is aggravated by the description of emerging resistance mechanisms, although with a polyclonal distribution profile.

The methicillin resistant Staphylococcus aureus (MRSA) is recognized by its ability to acquire and transferring resistance genes through interspecies conjugative plasmids. However, transference of plasmids from Gram-positive cocci to Gram-negative bacilli is not well characterized. In this report, we describe the transfer of a conjugative plasmid carrying qacA from MRSA to Escherichia coli C600. We performed a conjugation experiment using a chlorhexidine resistant MRSA isolate (ST-105/SCCmec type III) carrying the gene qacA and qacC as the donor and a chlorhexidine susceptible E. coli C600 isolate as the receptor. Transconjugants were selected using MacConkey agar plates containing chlorhexidine in concentrations ranging from 0.25 to 16 g.L-1. To genotypically confirm the transfer of the resistance gene, the transconjugants were screened by Polymerase Chain Reaction (PCR) and submitted to Sanger’s sequencing. MRSA isolates successfully transferred the chlorhexidine resistance gene (qacA) to the recipient E. coli strain C600. The E. coli transconjugant exhibited an important reduction of chlorhexidine susceptibility, with MICs increasing from ≤ 0.25 to ≥ 16 g.L-1 after conjugation. The qacA gene was detected by PCR as well as in the Sanger’s sequencing analysis of DNA from transconjugant plasmids. To the best of our knowledge, this is the first report of the plasmid p₈N_(q)ac(MN687830.1) carrying qacA and its transfer by conjugation from a MRSA to an E. coli. These findings increase concerns on the emergence of resistance dissemination across the genus and emphasizes the importance of continuous antiseptic stewardship.

Background: Assessing the risk of multidrug-resistant colonization and infections is pivotal for optimizing empirical therapy in hematopoietic stem cell transplants (HSCTs). Limited data exist on extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) colonization in this population. This study aimed to assess whether ESBL-E colonization constitutes a risk factor for ESBL-E bloodstream infection (BSI) and to evaluate ESBL-E colonization in HSCT recipients. Methods: A retrospective analysis of ESBL-E colonization and BSI in HSCT patients was conducted from August 2019 to June 2022. Weekly swabs were collected and cultured on chromogenic selective media, with PCR identifying the β-lactamase genes. Pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) assessed the colonizing strains’ similarities. Results: Of 222 evaluated HSCT patients, 59.45% were colonized by ESBL-E, with 48.4% at admission. The predominant β-lactamase genes were blaTEM (52%) and blaSHV (20%). PFGE analysis did not reveal predominant clusters in 26 E. coli and 15 K. pneumoniae strains. WGS identified ST16 and ST11 as the predominant sequence types among K. pneumoniae. Thirty-three patients developed thirty-five Enterobacterales-BSIs, with nine being third-generation cephalosporin-resistant. No association was found between ESBL-E colonization and ESBL-BSI (p = 0.087). Conclusions: Although the patients presented a high colonization rate of ESBL-E upon admission, no association between colonization and infection were found. Thus, it seems that ESBL screening is not a useful strategy to assess risk factors and guide therapy for ESBL-BSI in HSCT-patients.

Despite the widespread use of chlorhexidine (CHX) to prevent infection, data regarding the in vitro action of CHX against methicillin-resistant Staphylococcus aureus (MRSA) are limited. Clinical isolates from Hospital das Clinicas, Sao Paulo, Brazil, identified during 2002/2003 and 2012/2013 were studied to describe the susceptibility to CHX and mupirocin, molecular characteristics, and virulence profile of MRSA. Susceptibility test to Mupirocin was performed by the disk diffusion method and to CHX by the agar dilution technique. PCR for virulence genes, mecA gene and Staphylococcal Cassette Chromosome mec (SCCmec) types were investigated as well. Mupirocin- and CHX-resistant isolates were sequenced using the IlluminaTM plataform. Two hundred and sixteen MRSA clinical isolates were evaluated: 154 from infected and 62 from colonized patients. Resistance to mupirocin was observed in four isolates assigned as SCCmec type III and STs (ST05; ST239 and ST105) carrying mupA and blaZ, two of them co-harboring the ileS gene. Only one isolate assigned as SCCmec type III was resistant to CHX (MIC of 8.0 μg.mL-1) and harbored the qacA gene. Resistance to chlorhexidine and mupirocin were found in isolates carrying qacA and mupA in our hospital. Since these genes are plasmid-mediated, this finding draws attention to the potential spread of resistance to mupirocin in our hospital.

This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored blaKPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types.

The plasmid-mediated colistin-resistance gene named mcr-1 has been recently described in different countries and it became a public health challenge. Of note, few studies have addressed the spread of Escherichia coli harboring the mcr-1 gene in both, community and hospital settings. A total of seven colistin-resistant E. coli carrying mcr-1, collected from 2016 to 2018, from community (n=4), healthcare-acquired infections (n=2) and colonization (n=1) were identified in three high complexity hospitals in Sao Paulo, Brazil. These colistin-resistant isolates were screened for mcr genes by PCR and all strains were submitted to Whole Genome Sequencing and the conjugation experiment. The seven strains belonged to seven distinct sequence types (ST744, ST131, ST69, ST48, ST354, ST57, ST10), and they differ regarding the resistance profiles. Transference of mcr-1 by conjugation to E. coli strain C600 was possible in five of the seven isolates. The mcr-1 gene was found in plasmid types IncX4 or IncI2. Three of the isolates have ESBL-encoding genes (blaCTX-M-2, n=2; blaCTX-M-8, n=1). We hereby report genetically distinct E. coli isolates, belonging to seven STs, harboring the mcr-1 gene, associated to community and healthcare-acquired infections, and colonization in patients from three hospitals in Sao Paulo. These findings point out for the potential spread of plasmid-mediated colistin-resistance mechanism in E. coli strains in Brazil.