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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 (bla CTX-M-2, n=2; bla CTX-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.

Objectives Based on pulsed-field gel electrophoresis (PFGE) profile, whole-genome sequencing (WGS) of eight carbapenem-resistant Pseudomonas aeruginosa isolates from a bone marrow transplant unit in Sao Paulo, Brazil, was performed to investigate the presence of resistance and virulence genes as well as to determine the sequence type (ST) by multilocus sequence typing (MLST). Methods The initial phenotypic susceptibility pattern of the isolates was determined by VITEK®2. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method for amikacin, meropenem and colistin. WGS was performed using an Illumina MiSeq system. A Galleria mellonella infection model was used to evaluate the virulence of the strains. Results WGS demonstrated that mutations in genes encoding outer membrane proteins and efflux pumps in an isolate harbouring blaVIM-36 (ST308) differed from those in isolates harbouring blaSPM (ST277). The mexT gene harboured a mutation resulting in a frameshift in all isolates; in addition, the oprD gene of the blaVIM-36-carrying isolate had an insertion leading to a frameshift. Virulence genes did not differ between ST277 and ST308 strains. Moreover, only two isolates harbouring blaSPM showed virulence in the G. mellonella model, killing 100% of larvae after 18–24 h. Conclusions P. aeruginosa carrying blaVIM-36 belonging to ST308 was identified for the first time in our hospital. Although the virulence gene profiles were similar in isolates carrying blaSPM and the isolate carrying blaVIM-36, only two isolates harbouring blaSPM showed virulence in the G. mellonella model.

Purpose. Carbapenem resistance in P. aeruginosa is increasing worldwide. In Brazil, SPM-1 is the main P. aeruginosa carbapenemase identified. Little is known about the virulence factor in SPM-1 clones. Methodolgy. We describe a carbapenem-resistant P. aeruginosa bloodstream infection (CRPa-BSI) outbreak in a bone marrow transplant Unit (BMT). Twenty-nine CRPa-BSI cases were compared to 58 controls. Microbiological characteristics of isolates, such as sensitivity, carbapenemase gene PCR for P. aeruginosa, and PFGE are described, as well as the whole-genome sequence (WGS) of three strains. Results/Key findings. The cultures from environmental and healthcare workers were negative. Some isolates harboured KPC and SPM. The WGS showed that the 03 strains belonged to ST277, presented the same mutations in outer membrane protein, efflux pump, and virulence genes such as those involved in adhesion, biofilm, quorum-sensing and the type III secretion system, but differ regarding the carbapenemase profile. A predominant clone-producing SPM harbouring Tn 4371 was identified and showed cross-transmission; no common source was found. Overall mortality rate among cases was 79 %. The first multivariate analysis model showed that neutropenia (P=0.018), GVHD prophylaxis (P=0.016) and prior use of carbapenems (P=0.0089) were associated with CRPa-BSI. However, when MASCC>21 points and platelets were added in the final multivariate analysis, only prior use of carbapenems remained as an independent risk factor for CRPa-BSI (P=0.043). Conclusions. The predominant clone belonging to ST277 showed high mortality. Carbapenem use was the only risk factor associated with CRPa-BSI. This finding is a wake-up call for the need to improve management in BMT units.

Treatment of infections caused by A. baumannii is becoming a challenge due to the ability to develop multidrug-resistance, virulence, and high mortality. We described the colistin resistance and virulence genes present in sixA. baumannii clinical isolates using WGS, expression by qPCR, and virulence in the Galleria mellonella model. The colistin-resistant isolates were assigned as ST233 and the colistin-susceptible isolates as ST236 and ST407. The colistin-resistant isolates contained mutations within PmrA/PmrB, and the pmrA showed up-regulation in all of them. Only one colistin-resistant isolate indicating virulence in G. mellonella. This particular isolate belonged to a different clone, and it was the only isolate that presented non-synonymous mutations in pmrB. Colistinresistance in A. baumannii isolates seems to be caused by up-regulation of pmrA gene. Only one isolate appeared to be virulent in the G. mellonella model. This finding indicating low virulence in isolates belonging to emerging clones circulating in our hospital.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

We present a case report of a woman, concurrently colonized by polymyxin-resistant E. coli and K. pneumoniae. A Brazilian female patient, in her mid-fifties, was hospitalized with schistosomiasis. During hospitalization, polymyxin-resistant E. coli and K. pneumoniae were isolated from surveillance cultures. Identification, antimicrobial susceptibility testings, PCR for mcr-1, plasmid transfer by conjugation and whole genome sequencing were performed. E. coli ST744 and K. pneumoniae ST101 carrying mcr-1 gene were described. Transconjugant E. coli was positive for mcr-1 and IncX4 by PCR. The plasmid is a 33,304-base pair plasmid, and the mcr-1 gene was the only antimicrobial resistance gene present in the plasmid. This study presents a case report of a hospitalized woman, concurrently colonized by mcr-1-harboring E. coli ST744, a different ST from previously described in Brazil, and a K. pneumoniae ST101.

Background: Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important cause of nosocomial infections especially in intensive care units. This study aimed to assess clinical aspects and the genetic background of CRAb among ICU patients at a Brazilian teaching hospital. Methods: 56 critically ill patients colonized or infected by CRAb, during ICU stay, were prospectively assessed. Based on imipenem MIC ≥ 4 μg/mL, 28 CRAB strains were screened for the presence of genes encoding metallo-β-lactamases and OXA-type β-lactamases. The blaOXA-type genes were characterized by PCR using primers targeting ISAba-1 or -3. Genetic diversity of blaOXA-positive strains was determined by ERIC-PCR analysis. Results: Patient's mean age (±SD) was 61 (±15.1), and 58.9% were male. Eighty-percent of the patients presented risk factors for CRAb colonization, mainly invasive devices (87.5%) and previous antibiotic therapy (77.6%). Thirty-three patients died during hospital stay (59.0%). Resistance to carbapenems was associated with a high prevalence of blaOXA-23 (51.2%) and/or blaOXA-143 (18.6%) genes. ERIC-PCR genotyping identified 10 clusters among OXA-producing CRAb. Three CRAb strains exhibited additional resistance to polymyxin B (MIC ≥ 4 μg/mL), whereas 10 CRAb strains showed tigecycline MICs > 2 μg/mL. Conclusions: In this study, clonally unrelated OXA-123- and OXA-143-producing A. baumannii strains in ICU patients were strongly correlated to colonization with infected patients being associated with a poor outcome. Keywords: Acinetobacter baumannii, Carbapenem resistance, blaOXA-23, blaOXA-143

Pseudomonas aeruginosa is an important cause of infection, especially in immunocompromised patients. In this regard, strains producing carbapenemases, mainly metallo-β-lactamases (MBLs), have become a significant public health concern. Here, we present the complete annotated genome sequence (65.7 kb) of an F8-related lytic myovirus ( Pbunalikevirus genus) that infects MBL-producing P. aeruginosa strains.

This article is a revision of the previous edition article by Marjon H.J. Bennik, volume 3, pp 1867–1871, © 1999, Elsevier Ltd.

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We read with great interest C. S. Antonio et al.'s letter describing the high prevalence of Acinetobacter baumannii carrying bla OXA-143 in Brazilian hospitals ([1][1]). Recently, we carried out a similar study, and although the bla OXA-143 gene was identified, its frequency was lower than that

Pseudomonas aeruginosa (strain 48-1997A) producing Sao Paulo metallo-beta-lactamase (MBL)-1 (SPM-1) was described for the first time in 1997 and came from a 4-year-old leukemic girl in Sao Paulo, Brazil (15). Since then, SPM-1-producing P. aeruginosa has become endemic in Brazilian hospitals, being recurrently associated with outbreaks of nosocomial infection (8, 11, 12). More recently, the first reports of the MBL SPM-1 were described in Europe (14) and western Asia (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HM370523.1","term_id":"307949947","term_text":"HM370523.1"}}HM370523.1), denoting the potential for SPM-1 to become endemic worldwide (6). Unfortunately, carbapenem-resistant P. aeruginosa isolates carrying the SPM-1 enzyme have exhibited additional resistance to aminoglycosides, which has been mediated by a novel 16S rRNA methylase defined as RmtD (3), rendering ineffective a potent double-coverage regimen of a carbapenem plus an aminoglycoside and contributing to the emergence of panresistant phenotypes (3, 4). In this letter, we report the first environmentally isolated Pseudomonas aeruginosa carrying the blaSPM-1 and rmtD1 genes, providing additional data on the epidemiology of these genetic determinants of resistance. In August of 2010, during a surveillance study established to monitor the occurrence of antimicrobial resistance in Gram-negative bacteria from urban rivers in southeastern Brazil, a panresistant P. aeruginosa isolate (TIES-04900) was recovered from the Tiete River. Tiete is one of the main rivers of the region, which runs across Sao Paulo state from east to west for about 1,100 km. The water sample was collected from a dam located downstream of many important cities, including Sao Paulo, the largest and most populous metropolitan area in Brazil. P. aeruginosa strain TIES-04900 was resistant to imipenem, meropenem, ertapenem, ceftazidime, cefotaxime, cefepime, piperacillin-tazobactam, amikacin, tobramycin, gentamicin, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole; remaining susceptible only to aztreonam and polymyxin B (Table 1). MBL production was confirmed by using an MBL Etest strip. Based on the antimicrobial susceptibility pattern and genetic backgrounds of multidrug-resistant P. aeruginosa strains reported from Brazil (4, 8), antibiotic resistance genes were studied by PCR and sequencing, confirming the presence of the blaSPM-1, blaOXA-56, rmtD1, aacA4, aadA7, sul1, and dhfr genes (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"HQ876773","term_id":"323099809","term_text":"HQ876773"}}HQ876773 to {"type":"entrez-nucleotide","attrs":{"text":"HQ876777","term_id":"323099817","term_text":"HQ876777"}}HQ876777). This resistance genotype has been previously reported in clinical isolates of P. aeruginosa (3) and most likely has contributed to the endemicity of this panresistant phenotype in Brazilian hospitals (3, 4, 6, 8, 11, 12). In fact, enterobacterial repetitive intergenic consensus-PCR fingerprint analysis (Dice similarity coefficient and unweighted-pair group method using average linkages clustering), undertaken to ascertain the relatedness of TIES-04900 to clinical SPM-1- and RmtD-1-positive P. aeruginosa isolates (3, 15), showed that environmental P. aeruginosa TIES-04900 was clonally related to human P. aeruginosa strains PA0905 (95.2% similarity) and 48-1997A (90% similarity), previously identified in Sao Paulo (3, 15). Table 1. MICs of antibiotics for environmental Pseudomonas aeruginosa strain TIES-04900 carrying the blaSPM-1, blaOXA-56, rmtD1, aacA4, aadA7, sul1, and dhfr genesa In summary, we report the presence of SPM-1- and RmtD-1-coproducing P. aeruginosa in an urban river, showing that strains harboring the blaSPM-1 and rmtD1 genes seem not to be restricted to the hospital setting, a fact that emphasizes the importance of surveying environmental strains that might act as a source and/or reservoir of resistance genes with clinical relevance (1, 5, 7, 13) and have the potential for dissemination in communities through river environments (1, 10).

Balanoposthitis is defined as the inflammation of the glans penis and its foreskin. In the presence of other underlying medical conditions, this localized infection may spread systemically, serving as a source of fever and bacteremia in neutropenic males. Two rare cases of balanoposthitis caused by a clonally related Pseudomonas aeruginosa isolate co-producing the SPM-1 metallo-β-lactamase and the novel 16S rRNA methylase RmtD are described. Four multidrug-resistant (MDR) P. aeruginosa isolates were successively recovered from glans/foreskin swabs and urine cultures from two uncircumcised pediatric patients, one with Burkitt's non-Hodgkin's lymphoma and one with acute lymphoblastic leukemia. Clinically, preputial colonization by MDR P. aeruginosa evolved to severe balanoposthitis with glans/foreskin lesions as a source of fever. Combination therapy of ciprofloxacin and/or aztreonam (systemic) plus polymyxin B (topical) was effective once reversion of the neutropenic condition was achieved. Although P. aeruginosa remains an unusual cause of balanoposthitis, these cases should alert the physician to the potential pathogenicity of this bacterium. Furthermore, co-production of metallo-β-lactamase and 16S rRNA methylase has a potential impact on the empirical management of complicated infections caused by P. aeruginosa.

A recent article by Higgins et al. reported the identification of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL), defined as OXA-143, in a carbapenem-resistant Acinetobacter baumannii strain isolated in Brazil in 2004 (5). This enzyme, which has 88% identity with OXA-40, is the first representative of a novel subgroup of CHDLs whose prevalence remains to be determined (5). In an attempt to carry out this task, we have conducted a surveillance study of carbapenem-resistant A. baumannii isolates in order to determine the antimicrobial susceptibility patterns and prevalence of blaOXA-type carbapenemase genes in medical centers in both the southeastern and southern regions of Brazil. We report here a high prevalence of carbapenem-resistant A. baumannii carrying the blaOXA-143 and blaOXA-23 genes, along with the first isolation of strains carrying the blaOXA-72 (OXA-72 is a single-amino-acid variant of OXA-40) and blaOXA-58 genes in Brazilian hospitals. From 2004 to 2008, 36 carbapenem-resistant A. baumannii isolates recovered from different patients hospitalized in eight medical centers were screened for the presence of genes encoding metallo-β-lactamases (MβLs) and OXA-type β-lactamases (6, 15). Flanking sequences of blaOXA-type genes were characterized by PCR using primers targeting ISAba-1 or -3 (12). PCR products were confirmed by sequencing, and the genetic diversity of blaOXA-positive strains was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis (11). All isolates were found to be resistant to imipenem, meropenem, ceftazidime, aztreonam, piperacillin-tazobactam, and ciprofloxacin. Tobramycin (61.1%), ampicillin-sulbactam (61.1%), gentamicin (47.2%), amikacin (27.8%), and cefepime (11.1%) susceptibility rates were determined. MβL-encoding genes were not identified, whereas a high prevalence of blaOXA genes was noticed among carbapenem-resistant isolates (Table ​(Table1).1). In this regard, 21 strains (58.3%) carried the blaOXA-143 gene, 15 strains (41.7%) carried the ISAba-1/blaOXA-23 gene array, one strain carried the ISAba-3/blaOXA-58/ISAba-3 gene array, and two isolates carried the blaOXA-72 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ628170","term_id":"223585696","term_text":"FJ628170"}}FJ628170, {"type":"entrez-nucleotide","attrs":{"text":"FJ492877","term_id":"219563065","term_text":"FJ492877"}}FJ492877, {"type":"entrez-nucleotide","attrs":{"text":"FJ969387","term_id":"238866561","term_text":"FJ969387"}}FJ969387, and {"type":"entrez-nucleotide-range","attrs":{"text":"HM804278 to HM804281","start_term":"HM804278","end_term":"HM804281","start_term_id":"308737170","end_term_id":"308737176"}}HM804278 to HM804281). Although the presence of the blaOXA-51 and ISAba-1 genes was confirmed in all of the isolates, no colinearity of the two genes (i.e., ISAba-1 adjacent to the blaOXA-23 gene) was observed. Isolates harboring the blaOXA-143, blaOXA-72, and blaOXA-58 genes were restricted to hospitals located in Sao Paulo (the largest and most populous metropolitan area in southeastern Brazil), while blaOXA-23-harboring A. baumannii isolates were obtained from hospitals in Sao Paulo and Parana (in southern Brazil), confirming previous reports of widespread dissemination of OXA-23-producing A. baumannii in this region (2, 9). Finally, ERIC-PCR typing revealed genetic diversity among blaOXA-143- and blaOXA-23-positive A. baumannii isolates. TABLE 1. Characteristics of carbapenem-resistant A. baumannii isolates carrying blaOXA-type genes encoding class D carbapenemasesa In summary, the blaOXA-143 gene was commonly identified among the carbapenem-resistant A. baumannii isolates surveyed in this study. Also, we report the first isolation of blaOXA-72- and blaOXA-58-carrying strains in Brazilian hospitals. OXA-143 is a new subgroup of CHDL identified in isolates of A. baumannii from Brazil (5). We would only bring to mind that the blaOXA-72 gene was first reported from an A. baumannii strain in Thailand in 2004 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY739646","term_id":"52699554","term_text":"AY739646"}}AY739646), and so far it has been restricted to Acinetobacter sp. isolates from Asian and Mediterranean countries (1, 4, 7, 8, 14).