Co-carriage of Plasmid NDM and Chromosomal KPC in Klebsiella pneumoniae ST255 Human Wound Isolate in Brazil.

Multidrug-resistant K. pneumoniae is one of the main causes of hospital-acquired infections worldwide and frequently carries antimicrobial resistance genes in moving elements. In this study, we described a K. pneumoniae clinical isolate carrying simultaneous chromosomal blaKPC, and plasmid-mediated blaNDM and blaOXA-9. The isolate is multidrug-resistant and belongs to ST 225. While blaKPC were identified in the chromosome, the blaNDM was mediated by IncFII(K) plasmid and the blaOXA-9, in a IncFIB(K) plasmid. The blaKPC context was composed by Tn4401 transposon and two insertion sequences ISKpn6 and ISKpn7. The co-production of diverse ß-lactamases brings an alert about a new adaptive profile of K. pneumoniae strains and their dissemination in the hospital-acquired infectious.

Evaluation of XGEN Multi Sepsis Flow Chip Molecular Assay for Early Diagnosis of Bloodstream Infection.

Among healthcare-associated infections that can affect a critically ill patient, bloodstream infections are one of the most frequent causes of mortality, especially in hospitalized patients. The objective of this work is to evaluate the performance of the XGEN Multi Sepsis Flow Chip for the rapid diagnosis of bloodstream infections compared with conventional tests. In total, 101 positive blood culture samples were included, and the results obtained by the phenotypic conventional method (culture with susceptibility profile) were compared with results obtained by the XGEN Multi Sepsis Flow Chip. This molecular assay allows the simultaneous detection of the main bloodstream infection pathogens, and their most common antibiotic resistance markers in a short period of time. It was possible to observe substantial agreement between the methods for identifying the genus of pathogens. Considering species, the agreement was excellent. In relation to susceptibility, excellent agreement was noted between the detected resistance genes and susceptibility profile obtained through conventional antibiograms. The evaluated assay presented very early and satisfactory results for identification and detection of resistance genes of the main pathogens involved in bloodstream infections.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13 Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12 years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™ 2 × 300 bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

Broad-spectrum antimicrobial consumption trends and correlation with bacterial infections and antimicrobial resistance over 5 years.

OBJECTIVES: Consumption trends of four broad-spectrum antimicrobials and their correlation with antimicrobial resistance in Gram-negative bacilli (GNB) from 2013-2017 within intensive care units (ICUs) were explored. METHODS: Consumption of meropenem (MEM), polymyxin B (PMB), piperacillin/tazobactam (TZP) and cefepime (FEP) in defined daily doses per 1000 patient-days (DDD/1000PD) was measured. Infection-related GNB isolates were grouped according to specific resistance profiles. Time series of antimicrobial consumption and their parametric correlation with each grouped resistant GNB were explored. RESULTS: A total of 1423 GNB were evaluated. A significant linear decline in consumption was observed for MEM [slope -3.88, 95% confidence interval (CI) -4.96 to -2.81; P < 0.0001] and PMB (slope -3.51, 95% CI -5.528 to -1.495; P = 0.0009). A significant decline in MEM-non-susceptible Acinetobacter spp. (R2 = 0.476; P = 0.006) and an increase in FEP-non-susceptible Escherichia coli (R2 = 0.124; P = 0.006) was observed. A significant correlation between MEM consumption and MEM-non-susceptible Acinetobacter spp. (r = 0.43; P = 0.001) was observed. MEM consumption and MEM-non-susceptible Acinetobacter spp. showed a positive correlation. CONCLUSION: Reduction in the consumption of broad-spectrum antimicrobials may alter the frequency of infection-related isolates and their antimicrobial resistance profiles.

In vitro synergy of ticarcillin/clavulanate in combination with aztreonam and ceftolozane/tazobactam against SPM-1-producing Pseudomonas aeruginosa strains.

Minimal inhibitory concentrations (MICs) of ticarcillin/clavulanic acid (TLc), ceftolozane/tazobactam (C/T), and aztreonam (AT) were determined for 6 SPM-1-producing Pseudomonas aeruginosa (PSA) using Etest® strips and the synergistic effect of such antimicrobials against was evaluated by gradient diffusion strip crossing (GDSC) test. The fraction inhibitory concentration indexes (FICI) were calculated and showed a synergistic (n = 3) and additive (n = 2) effects of TLc + AT against SPM-1 producers, while TLc + C/T combination caused no effect. Average MIC reduction of TLc and AT by GDSC was 3-fold and 2-fold dilutions, respectively. Thus, TLc + AT might be a candidate as a combination therapy to treat SPM-1-producing PSA infections.

Dynamic of High-Risk Acinetobacter baumannii Major Clones in a Brazilian Tertiary Hospital During a Short Time Period.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 structure was found in the chromosome of five isolates, whereas blaOXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of ISAba1-blaADC-like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC50, > 512 mg/L) verified in all isolates. Only minocycline (MIC50, ≤ 0.5 µg/mL), polymyxin B (MIC50, 0.5 µg/mL), and tigecycline (MIC50, 0.5 µg/mL) were in vitro active against such isolates. A diversity of other antimicrobial resistance determinants (aph(3')-VIa, aadA1, aac(3')-IIa, strA, strB, sul2, drfA1, mph(E), msr(E), tetB, and floR) was also observed, which may confer resistance to at last six distinct antimicrobial classes. Four distinct pulsed-field gel electrophoresis (PFGE) profiles were observed during the study period, which belonged to ST79/ST258 (n = 2; IC5), ST25/ST229 (n = 2; IC7), ST1 (n = 1; IC1), and ST162/ST235 (n = 1; IC4). Although the ST1 isolate that carried blaOXA-23 and blaOXA-72 was introduced in this hospital setting by a transferred patient, two clonally related ST79/ST258 isolates carrying either one of these carbapenemase encoding genes were recovered from two patients who were hospitalized within the same period of time in the same hospital unit. Finally, a good correlation between PFGE/MLST, blaOXA-51 variant, and single nucleotide polymorphisms was also observed. Here we demonstrated that distinct extensively drug-resistant A. baumannii clones can circulate in the same hospital setting during a short time period, illustrating a very complex epidemiological scenario for this priority pathogen.

In vitro synergy of ceftolozane/tazobactam in combination with fosfomycin or aztreonam against MDR Pseudomonas aeruginosa.

OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-ß-lactamase production. This study evaluated in vitro antimicrobial synergy of ceftolozane/tazobactam in combination with aztreonam and fosfomycin against MDR PSA. METHODS: MICs were determined by broth microdilution and gradient strips. The effect of ceftolozane/tazobactam+aztreonam and ceftolozane/tazobactam+fosfomycin combinations were tested against 27 MDR PSA isolates carrying blaSPM-1 (n = 13), blaIMP (n = 4), blaVIM (n = 3), blaGES-1 (n = 2) and blaCTX-M-like (n = 2), and 3 isolates with no acquired ß-lactamase production detected by gradient diffusion strip crossing (GDSC). Six genetically unrelated SPM-1-producing isolates were also evaluated by time-kill analysis (TKA). RESULTS: All CR-PSA isolates harbouring blaSPM-1, blaGES-1 and blaIMP-1 were categorized as resistant to ceftolozane/tazobactam, meropenem and fosfomycin, with 70% being susceptible to aztreonam. Synergism for ceftolozane/tazobactam+fosfomycin and ceftolozane/tazobactam+aztreonam combinations was observed for 88.9% (24/27) and 18.5% (5/27) of the isolates by GDSC, respectively. A 3- to 9-fold reduction in ceftolozane/tazobactam MICs was observed, depending on the combination. Ceftolozane/tazobactam+fosfomycin was synergistic by TKA against one of six SPM-1-producing isolates, with additional non-synergistic bacterial density reduction for another isolate. Aztreonam peak concentrations alone demonstrated a ≥3 log10 cfu/mL reduction against all six isolates, but all strains were within the susceptible range for the drug. No antagonism was observed. CONCLUSIONS: In the context of increasing CR-PSA and the genetic diversity of resistance mechanisms, new combinations and stewardship strategies may need to be explored in the face of increasingly difficult to treat pathogens.

Gut Microbiota Differences in Children From Distinct Socioeconomic Levels Living in the Same Urban Area in Brazil.

OBJECTIVE: To compare gut microbiota in impoverished children versus children of high socioeconomic status living in the same urban area in Brazil. METHODS: A cross-sectional study was conducted to evaluate 100 children living in a slum and 30 children from a private school, ages between 5 and 11 years old, in Sao Paulo State, Brazil. To characterize the groups, data based on socioeconomic status, sanitation, and housing conditions were collected. Anthropometric measurements and neonatal data were obtained from both groups. Gut microbiota were quantified in fecal samples by real-time polymerase chain reaction. RESULTS: The children in the private school group had higher rates of cesarean delivery and premature birth than the children in the slum group. Staphylococcus aureus (90% vs 48.0%) and Clostridium difficile (100% vs 43.0%) were more commonly found in the children from the private school than in the impoverished children (P < 0.0001). C perfringens was most frequently identified in the group of children from the slum (92.0% vs 80%; P = 0.064). Higher counts of total eubacteria, Firmicutes and Bacteroidetes phyla organisms, Escherichia coli, Lactobacillus spp., and Methanobrevibacter smithii were found in the children living in poverty, whereas higher counts of Salmonella spp., C difficile, and C perfringens were observed in the children living in satisfactory housing conditions (P < 0.05). CONCLUSIONS: Important differences were observed between the gut microbiota of children living under distinct socioeconomic and environmental conditions within the same city. Our findings suggest that children of high socioeconomic status have less favorable gut microbiota than do children who live in poverty.

ß-Lactam Resistance Genes: Characterization, Epidemiology, and First Detection of blaCTX-M-1 and blaCTX-M-14 in Salmonella spp. Isolated from Poultry in Brazil-Brazil Ministry of Agriculture's Pathogen Reduction Program.

Salmonella spp. are widespread in nature; however, human infections occur mainly through ingestion of contaminated food, specially poultry and eggs. In Brazil, the Ministry of Agriculture (MAPA) oversees food production in general, with the goal of preventing transmission of pathogens through the food chain. In 2004, MAPA initiated a program to monitor and control levels of Salmonella in poultry during slaughter. This study analyzes isolates from MAPA's program for ß-lactam resistance and the resistance genes involved, as well as the geographic distributions of potentially clonal populations of resistant isolates within Brazil. Initially, 1,939 Salmonella spp. isolated between 2004 and 2011 were examined. These isolates were tested for antimicrobial susceptibility, and 100 isolates resistant or intermediate to ampicillin and ceftriaxone were screened initially for the presence of blaSHV, blaTEM, blaOXA, blaPSA, blaCMY-1, and blaCMY-2 genes. There were 55 isolates whose resistance genes were not identified by this panel and these isolates are the subject of this report. These 55 isolates were differentiated into 31 distinct ribogroups, with multiple ß-lactam resistance genes, including AmpC blaCMY, blaTEM, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, and blaCTX-M-14. Isolates carrying variants of blaCTX-M were identified in three geographic regions. Salmonella carrying particular genetic variants of blaCTX-M and belonging to the same ribogroup were identified from multiple poultry slaughtering facilities. In some instances, these presumptive clonal-related isolates were from facilities over 300 miles apart, indicating potential clonal spread between two geographic regions. This is the first report of blaCTX-M-1 and blaCTX-M-14 in Salmonella in Brazil.

Adhesion, biofilm and genotypic characteristics of antimicrobial resistant Escherichia coli isolates.

Aggregative adherence to human epithelial cells, most to renal proximal tubular (HK-2) cells, and biofilm formation was identified among antimicrobial resistant Escherichia coli strains mainly isolated from bacteremia. The importance of these virulence properties contributing to host colonization and infection associated with multiresistant E. coli should not be neglected.

Adhesion, biofilm and genotypic characteristics of antimicrobial resistant Escherichia coli isolates

Aggregative adherence to human epithelial cells, most to renal proximal tubular (HK-2) cells, and biofilm formation was identified among antimicrobial resistant Escherichia coli strains mainly isolated from bacteremia. The importance of these virulence properties contributing to host colonization and infection associated with multiresistant E. coli should not be neglected.

Differentiation of Klebsiella pneumoniae carbapenemase (KPC) variants by pyrosequencing.

A fast and reliable protocol using the pyrosequencing technique was developed to identify 11 different types of the KPC enzyme. A total of 65 blaKPC positive bacterial isolates were tested and characterized. In the end, the pyrosequencing proved to be a powerful tool for epidemiological studies of KPC producer isolates.

Characterization of ocular methicillin-resistant Staphylococcus epidermidis isolates belonging predominantly to clonal complex 2 subcluster II.

Staphylococcus epidermidis is an abundant member of the microbiota of the human skin and wet mucosa, which is commonly associated with sight-threatening infections in eyes with predisposing factors. Ocular S. epidermidis has become notorious because of its capability to form biofilms on different ocular devices and due to the evolving rates of antimicrobial resistance. In this study, the molecular epidemiology of 30 ocular methicillin-resistant S. epidermidis (MRSE) isolates was assessed using multilocus sequence typing (MLST). Antimicrobial resistance, accessory gene-regulator and staphylococcal cassette chromosome mec (SCCmec) types, biofilm formation, and the occurrence of biofilm-associated genes were correlated with MLST clonal complexes. Sequence types (STs) frequently found in the hospital setting were rarely found in our collection. Overall, 12 different STs were detected with a predominance of ST59 (30%), ST5 and ST6 (13.3% each). Most of the isolates (93.3%) belonged to the clonal complex 2 (CC2) and grouped mainly within subcluster CC2-II (92.9%). Isolates grouped within this subcluster were frequently biofilm producers (92.3%) with a higher occurrence of the aap (84.5%) and bhp (46.1%) genes compared to icaA (19.2%). SCCmec type IV (53.8%) was predominant within CC2-II strains, while 38.4% were nontypeable. In addition, CC2-II strains were frequently multidrug resistant (80.7%) and demonstrated to be particularly resistant to ciprofloxacin (80.8%), ofloxacin (77%), azithromycin (61.5%), and gentamicin (57.7%). Our findings demonstrate the predominance of a particular MRSE cluster causing ocular infections, which was associated with high rates of antimicrobial resistance and particularly the carriage of biofilm-related genes coding for proteinaceous factors implicated in biofilm accumulation.

Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections.

INTRODUCTION: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. METHODS: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. RESULTS: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsule-producing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. CONCLUSIONS: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients' isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome.

Rapid detection of carbapenemase genes by multiplex real-time PCR.

OBJECTIVES: To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). METHODS: A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). RESULTS: Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. CONCLUSIONS: The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

A spatial approach for the epidemiology of antibiotic use and resistance in community-based studies: the emergence of urban clusters of Escherichia coli quinolone resistance in Sao Paulo, Brasil.

BACKGROUND: Population antimicrobial use may influence resistance emergence. Resistance is an ecological phenomenon due to potential transmissibility. We investigated spatial and temporal patterns of ciprofloxacin (CIP) population consumption related to E. coli resistance emergence and dissemination in a major Brazilian city. A total of 4,372 urinary tract infection E. coli cases, with 723 CIP resistant, were identified in 2002 from two outpatient centres. Cases were address geocoded in a digital map. Raw CIP consumption data was transformed into usage density in DDDs by CIP selling points influence zones determination. A stochastic model coupled with a Geographical Information System was applied for relating resistance and usage density and for detecting city areas of high/low resistance risk. RESULTS: E. coli CIP resistant cluster emergence was detected and significantly related to usage density at a level of 5 to 9 CIP DDDs. There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. CONCLUSIONS: There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. The usage density of 5-9 CIP DDDs per 1,000 inhabitants within the same influence zone was the resistance triggering level. This level led to E. coli resistance clustering, proving that individual resistance emergence and dissemination was affected by antimicrobial population consumption.

First description of bla(CTX-M-14)- and bla(CTX-M-15)-producing Escherichia coli isolates in Brazil.

We evaluated the antimicrobial resistance patterns and molecular characteristics of 11 extraintestinal Escherichia coli strains and 1 intestinal E. coli from human infections collected in Brazil. Two E. coli strains were nonsusceptible to extended spectrum cephalosporins (cefotaxime, ceftazidime, and cefepime); one isolated from diarrhea carried bla(CTX-M-14) and bla(TEM-1), whereas the other, isolated from tracheal secretion, carried bla(CTX-M-15) and bla(OXA-1). Five E. coli strains showed resistance to quinolones. Integrase associated with class 1 integron (intl1) was detected in 8 of the 12 E. coli strains belonging to various serotypes and this gene was carried by plasmids showing similar size. Pulsed-field gel electrophoresis showed that E. coli strains were genetically diverse, and phylogenetic grouping showed that the E. coli strains belonged to groups A, B2, and D (33.3%), respectively. This is the first report of E. coli isolates carrying bla(CTX-M-14) and bla(CTX-M-15) in Brazil. The presence of mobile elements containing antimicrobial resistance genes is worrisome since it could promote the dissemination of resistance and lead to the acquisition of resistance to other antimicrobials agents such as the carbapenems.