Rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing Klebsiella pneumoniae.

Enterobacteriaceae strains producing the Klebsiella pneumoniae carbapenemase (KPC) have disseminated worldwide, causing an urgent threat to public health. KPC-producing strains often exhibit low-level carbapenem resistance, which may be missed by automated clinical detection systems. In this study, eight Klebsiella pneumoniae strains with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility to high-level resistance as defined by clinical laboratory testing standards. Time-kill analysis with an inoculum as low as 3 × 10(6) CFU/ml and concentrations of imipenem 8- and 16-fold higher than the MIC resulted in the initial killing of 99.9% of the population. However, full recovery of the population occurred by 20 h of incubation in the same drug concentrations. Population profiles showed that recovery was mediated by a heteroresistant subpopulation at a frequency of 2 × 10(-7) to 3 × 10(-6). Samples selected 2 h after exposure to imipenem were as susceptible as the unexposed parental strain and produced the major outer membrane porin OmpK36. However, between 4 to 8 h after exposure, OmpK36 became absent, and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 h of exposure revealed both susceptible and resistant subpopulations. Once induced, however, the high-level imipenem resistance was maintained, and OmpK36 remained unexpressed even without continued carbapenem exposure. This study demonstrates the essential coordination between blaKPC and ompK36 expression mediating high-level imipenem resistance from a population of bacteria that initially exhibits a carbapenem-susceptibility phenotype.

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-ß-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 µg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.

Ciprofloxacin prophylaxis in high risk neutropenic patients: effects on outcomes, antimicrobial therapy and resistance.

BACKGROUND: The use of quinolone prophylaxis in high-risk neutropenic patients is considered standard of care but the development of resistance is a concern. Previous studies have focused mainly on quinolone resistance among patients receiving prophylaxis, with very few data reporting its impact on the hospital microbial epidemiology. METHODS: We analyzed a cohort of 329 episodes of chemotherapy-induced neutropenia in adults, and compared two periods: 2005 (period 1, no prophylaxis, n=110) and 2006-2008 (period 2, ciprofloxacin prophylaxis, n=219). Outcomes analyzed were the frequency of febrile neutropenia, bacteremia, duration of antibiotic therapy and hospitalization, and antimicrobial resistance to ciprofloxacin and extended-spectrum beta-lactamase [ESBL] production. We analyzed resistance rates (by patients-day) in the cohort, as well as in other patients (neutropenic and non-neutropenic, 11,975 patients-day) admitted to the hematology unit in the same period, taking into consideration the general resistance patterns in the hospital. RESULTS: Quinolone prophylaxis (period 2) resulted in fewer episodes of febrile neutropenia (159/219 [73%] vs. 102/110 [93%], Chi-square 18.09, p = 0.00002), and bacteremia (49/219 [22] vs. 36/110 [33%], Chi-square 4.10, p = 0.04), shorter duration of antibiotic therapy (p = 0.0002) and hospitalization (p = 0.002), but more frequent use of carbapenems (79/219 [36%] vs. 15/110 [14%], Chi-square 18.06, p = 0.0002). In addition, period 2 was associated with higher rates of quinolone resistance (6.77 vs. 3.02 per 1,000 patients-day, p = 0.03). The rate of ESBL-producing enterobacteria in the two periods was slightly higher in patients receiving quinolone prophylaxis (1.27 vs. 0.38 per 1,000 patients-day, p = 0.26) as well as in the hematology unit overall (1.59 vs. 0.53 per 1,000 patients-day, p = 0.08), but remained stable in the whole hospital (0.53 vs. 0.56 per 1,000 patients-day, p = 0.74). CONCLUSIONS: Ciprofloxacin prophylaxis was beneficial in high risk neutropenic patients, but important modifications in the prescription of carbapenems and on antimicrobial resistance patterns of isolates were observed. The importance of hospital or ward ecology must be taken into account when deciding for quinolone prophylaxis in high-risk neutropenic patients.

Use of fimH single-nucleotide polymorphisms for strain typing of clinical isolates of Escherichia coli for epidemiologic investigation.

Strain typing methods that compare electrophoresis banding patterns are commonly used but are difficult to standardize and poorly portable. Multilocus sequence typing (MLST) is a sequence-based alternative, but it is not practical for large-scale epidemiological studies. In the present study, the usefulness of fimH single-nucleotide polymorphisms (SNPs) for Escherichia coli typing was explored. fimH SNPs were determined for 345 E. coli clinical isolates (including 3 reference strains) and compared to PCR-based ECOR (E. coli reference collection) phylogrouping. The fimH gene could be amplified for 316 (92%) of the 345 isolates. fimH SNP analysis found 46 distinct terminal groups in the nucleotide sequence-based phylogenetic tree (fimH types). A subset of the E. coli isolates (162 clinical isolates and the 3 reference strains) were compared by fimH type, PCR phylogroup, and MLST. These isolates fell into 27 fimH types and 18 MLST clonal complexes (CCs) that contained 2 to 28 isolates per complex. The combination of PCR phylogroup and fimH type corresponded to a single CC for 113 (68%) isolates and 2 or 3 CCs for the other 52 (32%) isolates. We propose that the combination of PCR phylogrouping and fimH SNP analysis may be a useful method to type a large collection of clinical E. coli isolates for epidemiologic studies.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients.

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.

Colistin resistance emerges in pandrug-resistant Klebsiella pneumoniae epidemic clones in Rio de Janeiro, Brazil.

Klebsiella pneumoniae is an important human pathogen, able to accumulate and disseminate a variety of antimicrobial resistance genes. Resistance to colistin, one of the last therapeutic options for multi-drug-resistant bacteria, has been reported increasingly. Colistin-resistant K. pneumoniae (ColRKp) emerged in two hospitals in Rio de Janeiro state, Brazil in 2016. The aim of this study was to investigate if these ColRKp isolates were clonally related when compared between hospitals, to identify the molecular mechanisms of colistin resistance, and to describe other antimicrobial resistance genes carried by isolates. Twenty-three isolates were successively recovered, and the whole-genome sequence was analysed for 10, each of a different pulsed-field gel electrophoresis (PFGE) type. Although some PFGE clusters were found, none of them included isolates from both hospitals. Half of the isolates were assigned to CC258, three to ST152 and two to ST15. One isolate was pandrug resistant, one was extensively drug resistant, and the others were multi-drug resistant. Colistin resistance was related to mutations in mgrB, pmrB, phoQ and crrB. Eleven new mutations were found in these genes, including two nucleotide deletions in mgrB. All isolates were carbapenem resistant, and seven were associated with carbapenemase carriage (blaKPC-2 in six isolates and blaOXA-370 in one isolate). All isolates had a blaCTX-M, and two had a 16S ribosomal RNA methyltransferase encoding gene (armA and rmtB). ColRKp were composed of epidemic clones, but cross-dissemination between hospitals was not detected. Colistin resistance emerged with several novel mutations amid highly resistant strains, further restricting the number of drugs available and leading to pandrug resistance.

A carbapenem-susceptible Pseudomonas aeruginosa strain carrying the bla(SPM) gene.

We studied a carbapenem-susceptible Pseudomonas aeruginosa strain that does not produce carbapenemase but carries the metallo-beta-lactamase gene bla(SPM) identical in sequence to the gene of other fully carbapenem-resistant isolates. Carbapenem-susceptible isolates may be silent reservoirs of the bla(SPM) gene.

Draft Genome Sequence of a Tetracycline-Resistant Plesiomonas shigelloides Strain Isolated from Aquaculture-Reared Tilapia.

We hereby present the 3.7-Mb draft genome sequence of Plesiomonas shigelloides strain FM82, isolated from a tilapia (Oreochromis niloticus) reared in a fish farm in Rio de Janeiro, Brazil. P. shigelloides strain FM82 carries antimicrobial resistance, biofilm, and CRISPR-related genes.

Multilocus sequence typing of Staphylococcus aureus isolates recovered from cows with mastitis in Brazilian dairy herds.

Staphylococcus aureus is a major pathogen associated with bovine mastitis, one of the most important infectious diseases occurring in dairy cattle herds worldwide. In the present study, S. aureus isolates recovered from cows with mastitis in dairy herds located in the south-east of Brazil were genotyped by PFGE and multilocus sequence typing (MLST). PFGE identified 60 pulsotypes (PTs), which were found to be distributed among six clonal complexes (CCs) by MLST. All PTs with similarity percentages greater than 65 % belonged to the same CC. Most of the PTs belonged to CC126 (n=28) and CC97 (n=19), which were represented by 91 % of the isolates. These CCs have also been recovered from cows with mastitis in countries located in different continents, but they have rarely been isolated from human specimens. Few isolates were represented by PTs belonging to CCs that are frequently isolated from human specimens (CC1, CC5 and CC30). These data reinforce the hypothesis that a limited number of S. aureus CCs are responsible for most bovine mastitis cases internationally. Specific features of the specialized clones should be studied for use as future targets of mastitis control measures.

Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples.

The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false-positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

BlaGES carrying Pseudomonas aeruginosa isolates from a public hospital in Rio de Janeiro, Brazil.

Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.

Mupirocin for controlling methicillin-resistant Staphylococcus aureus: lessons from a decade of use at a university hospital.

BACKGROUND: From 1990 to 1995 at Hospital Universitário Clementino Fraga Filho, patients colonized or infected with methicillin-resistant Staphylococcus aureus (MRSA) were treated with mupirocin to eliminate MRSA carriage. In 1995, 65% of MRSA patients at this hospital had mupirocin-resistant isolates. Starting in 1996, mupirocin use was restricted to patients colonized, but not infected, with MRSA. OBJECTIVES: To describe the use of mupirocin for controlling MRSA over a decade and to analyze the molecular epidemiology of mupirocin-resistant MRSA infections at this hospital. SETTING: A 490-bed, tertiary-care university hospital. METHODS: The incidence densities of patients with MRSA and acquisition of mupirocin by the hospital were calculated for the period 1992-2001. S. aureus isolates from 1999-2000 were analyzed by pulsed-field gel electrophoresis. Mupirocin-resistant MRSA isolates from 1994-1995 and 1999-2000 were analyzed for ileS-2 gene background polymorphisms. RESULTS: The incidence density of MRSA patients increased slightly over time, whereas the purchase of mupirocin decreased dramatically. Mupirocin-resistant MRSA infections decreased from 65% in 1994-1995 to 15% in 1999-2000. The MRSA Brazilian clone, detected in 1992, was still highly prevalent. The same ileS-2 encoding plasmid found in 1994-1995 persisted in three identical MRSA isolates from 1999-2000 belonging to the Brazilian clone. CONCLUSIONS: After mupirocin use decreased, the ileS-2 encoding plasmid persisted in only a few Brazilian clone isolates. Our data on mupirocin-resistant MRSA incidence and mupirocin use strongly suggested that restricted use was related to decreased rates of mupirocin resistance at our hospital.

Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples

Abstract The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

Clonal composition of Escherichia coli causing community-acquired urinary tract infections in the State of Rio de Janeiro, Brazil.

Recent studies from North America and Europe have demonstrated community-wide clonal spread of uropathogenic Escherichia coli (UPEC). To investigate if a similar pattern of spread occurs in Brazil, we characterized UPEC from women with community-acquired urinary tract infection (UTI) in Rio de Janeiro. E. coli isolates from women with UTI in one public outpatient clinic were evaluated for antibiotic susceptibility, E. coli phylogenetic grouping, enterobacterial repetitive intergenic consensus (ERIC) 2 PCR and pulsed-field gel electrophoresis fingerprinting, and multilocus sequence typing. From March 2005 to November 2006, 344 patients were studied. Of these, 186 (54%) had confirmed UTI, 118 (63.4%) of which were caused by E. coli. More than 50% of these isolates were resistant to ampicillin and trimethoprim/sulfamethoxazole. Of these, 96 (81%) belonged to 19 ERIC2 clonal groups. The largest group included 15 isolates, all belonging to multilocus sequence typing group ST69 and phylogenetic group D; they had pulsed-field gel electrophoresis patterns sharing at least 89% similarity compared with the CgA reference strain ATCC BAA-457. CgA strains have been found to be widespread in the United States in the early 2000s. Clonal group E. coli strains accounted for a large proportion (52%) of all UTIs and 82% of the trimethoprim/sulfamethoxazole-resistant E. coli UTIs. Thus, as in North America and Europe, UPECs that cause UTI in Rio de Janeiro also show clonal distribution, and a substantial proportion of drug-resistant UTI is caused by a small set of genetically related E. coli strains.

Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages.

In only a few instances has the clonal composition of Staphylococcus aureus collections that include methicillin-susceptible S. aureus (MSSA) been extensively characterized. In order to investigate the clonal composition of MSSA and methicillin-resistant S. aureus (MRSA) and examine whether the infections diagnosed at our hospital were related to internationally distributed S. aureus lineages, we collected 89 clinical S. aureus isolates from patients at a public university hospital in Rio de Janeiro, Brazil, from September 1999 to June 2000. All S. aureus isolates were genotyped by pulsed-field gel electrophoresis and multilocus restriction fragment typing (MLRFT), and a subset (n = 17) was further characterized by multilocus sequence typing (MLST). The 34 MRSA isolates were additionally characterized by SCCmec typing. The MSSA population (n = 55) was grouped into 18 restriction fragment types (RFTs); of these, five RFTs accounted for 67% (37) of the MSSA isolates. MRSA isolates were clustered into only three RFTs (P = 0.02). The majority of MSSA RFTs were related to sequence type 30 (ST30) (12 isolates, 22%), ST1, ST188, and ST432 (6 isolates, 11% each). The predominant MRSA RFT comprised 31 (91%) of 34 isolates; four randomly selected isolates of this RFT were ST239, the previously described widely disseminated Brazilian clone. However, a fifth isolate belonging to this RFT was the ST644, a new single locus variant of ST239. By applying MLRFT and MLST, we found evidence for a clonal structure in MSSA isolates and detected the dissemination of MSSA clonal complexes 1, 5, 8, 30, and 45.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.

blaGES carrying Pseudomonas aeruginosa isolates from a public hospital in Rio de Janeiro, Brazil

Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.