Description of a new non-Tn4401 element (NTEKPC-IIe) harboured on IncQ plasmid in Citrobacter werkmanii from recreational coastal water.

OBJECTIVES: Here we describe an IncQ1-like plasmid carrying blaKPC-2 in a new non-Tn4401 element found in Citrobacter werkmanii recovered from coastal water. METHODS: In vitro and in silico approaches were used to assess antimicrobial resistance determinants, as well as blaKPC-2 vicinities. RESULTS: The LB-887 isolate showed a multidrug-resistant phenotype and was identified as C. werkmanii. Resistome analysis identified further acquired resistance determinants to ß-lactams, aminoglycosides, sulphonamides/trimethoprim, tetracyclines, chloramphenicol, macrolides, rifampicin and fluoroquinolones. Plasmidome included incompatibility groups IncA, IncC2, IncR, Col and IncQ families. The blaKPC-2 was inserted on a new variant of NTEKPC-II, called here NTEKPC-IIe, carried by an InQ1-like plasmid of 7930 kb (pKPC-LB887). NTEKPC-IIe differed from NTEKPC-IId by the complete absence of ISKpn6-tnpA. The InQ1-like backbone harbouring this element had been described in Enterobacterales recovered from clinical and environmental settings. CONCLUSION: Unravelling genetic structures related to blaKPC dissemination in different settings may provide clues on the main forces driving evolution of this important resistance determinant. Indeed, the occurrence of blaKPC in a new NTEKPC variant from an environmental source highlights the ongoing evolution of this mobile genetic element. In addition, blaKPC carriage on a small and highly mobilizable IncQ plasmid in C. freundii complex from recreational water, similar to others found in clinical isolates, may suggest its relevance for blaKPC-2 dissemination among different compartments.

Comparative Genomics of Acinetobacter baumannii Clinical Strains From Brazil Reveals Polyclonal Dissemination and Selective Exchange of Mobile Genetic Elements Associated With Resistance Genes.

Acinetobacter baumannii is an opportunistic bacterial pathogen infecting immunocompromised patients and has gained attention worldwide due to its increased antimicrobial resistance. Here, we report a comparative whole-genome sequencing and analysis coupled with an assessment of antibiotic resistance of 46 Acinetobacter strains (45 A. baumannii plus one Acinetobacter nosocomialis) originated from five hospitals from the city of Recife, Brazil, between 2010 and 2014. An average of 3,809 genes were identified per genome, although only 2,006 genes were single copy orthologs or core genes conserved across all sequenced strains, with an average of 42 new genes found per strain. We evaluated genetic distance through a phylogenetic analysis and MLST as well as the presence of antibiotic resistance genes, virulence markers and mobile genetic elements (MGE). The phylogenetic analysis recovered distinct monophyletic A. baumannii groups corresponding to five known (ST1, ST15, ST25, ST79, and ST113) and one novel ST (ST881, related to ST1). A large number of ST specific genes were found, with the ST79 strains having the largest number of genes in common that were missing from the other STs. Multiple genes associated with resistance to ß-lactams, aminoglycosides and other antibiotics were found. Some of those were clearly mapped to defined MGEs and an analysis of those revealed known elements as well as a novel Tn7-Tn3 transposon with a clear ST specific distribution. An association of selected resistance/virulence markers with specific STs was indeed observed, as well as the recent spread of the OXA-253 carbapenemase encoding gene. Virulence genes associated with the synthesis of the capsular antigens were noticeably more variable in the ST113 and ST79 strains. Indeed, several resistance and virulence genes were common to the ST79 and ST113 strains only, despite a greater genetic distance between them, suggesting common means of genetic exchange. Our comparative analysis reveals the spread of multiple STs and the genomic plasticity of A. baumannii from different hospitals in a single metropolitan area. It also highlights differences in the spread of resistance markers and other MGEs between the investigated STs, impacting on the monitoring and treatment of Acinetobacter in the ongoing and future outbreaks.

Rapid Detection of Echinocandins Resistance by MALDI-TOF MS in Candida parapsilosis Complex.

Mass spectrometry by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was used to identify and differentiate the pattern of susceptibility of clinical isolates of Candida parapsilosis complex. 17 C. parapsilosis sensu stricto, 2 C. orthopsilosis, and 1 C. metapsilosis strains were obtained from blood cultures, and three different inocula (103, 105, and 107 CFU/mL) were evaluated against three echinocandins at concentrations ranging from 0.03 to 16 µg/mL after incubation of 1 h, 2 h, and 3 h. Drug-free control was used. The spectra obtained at these concentrations were applied to generate composite correlation index (CCI) matrices for each yeast individually. After cross correlations and autocorrelations of each spectra with null (zero) and maximal (16) concentrations, the CCI was used as separation parameter among spectra. Incubation time and inoculum were critical factors to reach higher precision and reliability of this trial. With an incubation time of 3 h and inoculum of 107 CFU/mL, it was possible to determine the breakpoint of the clinical yeasts by MALDI-TOF that presented high agreement with the clinical laboratory standard institute (CLSI) reference method. Herein, we show that mass spectrometry using the MALDI-TOF technique is powerful when it exploits antifungal susceptibility testing assays.

Identification of Lactic Acid Bacteria in Fruit Pulp Processing Byproducts and Potential Probiotic Properties of Selected Lactobacillus Strains.

This study aimed to identify lactic acid bacteria (LAB) in byproducts of fruit (Malpighia glabra L., Mangifera indica L., Annona muricata L., and Fragaria vesca L.) pulp processing. Fifty strains of LAB were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence (16S rRNA) analysis. Species belonging to Lactobacillus genus were the predominant LAB in all fruit pulp processing byproducts. The average congruency between the MALDI-TOF MS and 16S rRNA in LAB species identification reached 86%. Isolates of L. plantarum, L. brevis, L. pentosus, L. lactis and L. mesenteroides were identified with 100% congruency. MALDI-TOF MS and 16S rRNA analysis presented 86 and 100% efficiency of LAB species identification, respectively. Further, five selected Lactobacillus strains (L. brevis 59, L. pentosus 129, L. paracasei 108, L. plantarum 49, and L. fermentum 111) were evaluated for desirable probiotic-related properties and growth behavior on two different cultivation media. The exposure to pH 2.0 sharply decreased the counts of the different Lactobacillus strains after a 1 or 2 h incubation, while varied decreases were noted after 3 h of exposure to pH 3.0. Overall, the exposure to pH 5.0 and to bile salts (0.15, 0.30, and 1.00%) did not decrease the counts of the Lactobacillus strains. All tested Lactobacillus strains presented inhibitory activity against Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli, and presented variable susceptibility to different antibiotics. The selected Lactobacillus strains presented satisfactory and reproducible growth behavior. In conclusion, MALDI-TOF MS and 16S rRNA analysis revealed high efficiency and congruency for LAB species identification, and the selected Lactobacillus strains may be candidates for further investigation of novel probiotic strains.

Comparative analysis of the complete genome of KPC-2-producing Klebsiella pneumoniae Kp13 reveals remarkable genome plasticity and a wide repertoire of virulence and resistance mechanisms.

BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342. RESULTS: We have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains. CONCLUSIONS: Our results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Beta-lactam resistance mechanisms in Pseudomonas aeruginosa strains causing bloodstream infections: comparative results between Brazilian and American isolates.

This study evaluated the presence of distinct mechanisms of beta-lactam resistance in 122 Pseudomonas aeruginosa isolates, causing bloodstream infections at Hospital São Paulo (HSP, Brazil; 82 isolates) and Virginia Commonwealth University Medical Center (VCU, United States; 40 isolates). By Clinical Laboratory Standards Institute agar dilution, Brazilian P. aeruginosa isolates showed higher resistance rates to most antimicrobials tested than those collected from the United States, except for ciprofloxacin. Carbapenem hydrolysis was detected in seven P. aeruginosa from HSP, in which bla(SPM-1) (n=5), bla(IMP-1) (n=1), and bla(IMP-16) (n=1) were detected by polymerase chain reaction (PCR) followed by DNA sequencing. The production of GES-5 was observed in 1.25% of HSP isolates. No extended-spectrum beta-lactamase-encoding genes were detected in the VCU isolates. Expression of efflux systems genes (mexB, mexD, mexF, and mexY) was evaluated by quantitative reverse transcriptase-PCR. In HSP isolates MexXY-OprM (41.4%) efflux system was more frequently overexpressed, in contrast to what was observed in the VCU isolates, where both MexXY-OprM (25.0%) and MexAB-OprM (25.0%) were equally overexpressed. The oprD downregulation was similar among isolates collected from the HSP (92.7%) and VCU (95.0%). On the other hand, ampC overexpression was observed only among HSP isolates (31.7%). The distinct antimicrobial susceptibility profile and mechanisms of beta-lactam resistance found among P. aeruginosa isolated from teaching hospitals located in Brazil and the United States exemplify the importance of local epidemiology in determining antimicrobial resistance rates.

Efflux pumps expression and its association with porin down-regulation and beta-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil.

BACKGROUND: Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. RESULTS: Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC beta-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. CONCLUSIONS: Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary beta-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates.

Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results.

OBJECTIVES: The aim of this study was to evaluate the presence of carbapenemases in a Klebsiella pneumoniae collection and the performance of the modified Hodge test (MHT) to correctly identify this phenotype. METHODS: Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS-PAGE. RESULTS: Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla(CTX-M) in 27 (96%) isolates, of which 2 isolates also carried bla(GES-1.) SDS-PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36. CONCLUSIONS: False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum beta-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.