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We describe a cross-sectional survey to identify risk factors for colonisation of neonates by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae. This occurred following exposure to a colonised healthcare worker during an outbreak in an intermediate-risk neonatal unit. In total, 120 neonates admitted consecutively during a three-month period were screened for ESBL-producing K. pneumoniae by rectal swabbing and 27 were identified as colonised. Multivariate analysis showed colonisation to be independently associated with use of antibiotics and absence of breastfeeding. Previous use of antibiotics presented an odds ratio (OR) of 12.3 [95% confidence interval (CI): 3.66-41.2, P<0.001]. The most commonly used antibiotics were penicillin and amikacin. Breastfeeding was associated with reduced risk for colonisation (OR: 0.22; 95% CI: 0.05-0.99; P=0.049). Nine isolates recovered during the first stage of the outbreak and 27 isolates from surveillance cultures were typed thereafter by pulsed-field gel electrophoresis, revealing six different profiles (A-F). Clones A, C, and E were implicated in the first stage of the outbreak, whereas among the 27 strains recovered from surveillance cultures, all six clones were identified. Clone A was also found on the hand of a nursing auxiliary with onychomycosis. We concluded that prior antimicrobial use predisposed to colonisation. The possible role of breastfeeding as a protective factor needs to be further elucidated. Detection of different genotypes of ESBL-producing K. pneumoniae suggests that dissemination of mobile genetic elements bearing the ESBL gene may have been superimposed on the simple dissemination of a clone during the outbreak. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin., Methods: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp. IEC33019 strain. Southern blotting/hybridization assays were performed to confirm the location of the MBL-encoding gene, blaBIM-1 (Belém Imipenemase). Antimicrobial susceptibility testing, cloning, and biochemical and phenotypic characterization were performed to determine BIM-1 kinetics., Results: The IEC33019 strain showed high resistance rates to β-lactams, ciprofloxacin and aminoglycosides, being susceptible only to polymyxins and susceptible, increased exposure to aztreonam. WGS analysis revealed a novel acquired MBL-encoding gene, blaBIM-1, found as a gene cassette inserted into a class 1 integron (In1326) that also carried qnrVC1 and aadA11e. In1326 was located in a complex transposon, Tn7122, carried by a 52.7 kb conjugative plasmid (pIEC33019) with a toxin/antitoxin system (vapB/vapC). BIM-1 belongs to the molecular subgroup B1 and shares 70.2% and 64.9% similarity with SIM-1 and IMP-1, respectively. Kinetics analysis of BIM-1 showed hydrolytic activity against all β-lactams tested., Conclusions: BIM-1 is a novel acquired MBL encoded by a gene carried by mobile genetic elements, which can be transferred to other Gram-negative bacilli (GNB). Because the IEC33019 strain was recovered from a river impacted by a populous metropolitan region with poor basic sanitation and served by limited potable freshwater, it would be important to establish the role of the BIM-1-producing GNB as nosocomial pathogens and/or as colonizers of the riverside population in this geographical region., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Introduction: Staphylococcus aureus may provoke peritonitis and death, especially in immunocompromized individuals such as diabetic patients. We evaluated the role of insulin in S. aureus -induced peritoneal infection in diabetic and non-diabetic rats. Materials/Methods: Alloxan-diabetic male Wistar rats and their respective controls received intraperitoneal injections of different strains of S. aureus or sterile phosphate-buffered saline. After 3 days of infection, the first set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of neutral protamine Hagedorn insulin and were analyzed 8 h later. The second set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of insulin 2 h before intraperitoneal infection and a half dose of insulin at 5 p.m. for the next 2 days and were analyzed 16 h later. The following measurements were performed: (a) number of cells in the peritoneal lavage fluid (PeLF), white blood cell count, and blood glucose; (b) serum insulin and corticosterone; (c) cytokine levels in the PeLF; (d) expression of adhesion molecules in the vascular endothelium; and (e) microbicidal activity. Results: Diabetic rats showed an increased number of polymorphonuclear leukocytes (PMNs) and increased concentrations of CINC-1, IL-4, and IFN-γ in the PeLF after infection with the ATCC 25923 or N315 αHL + strain. The mesenteric expression of PECAM-1 was increased after infection with the N315 HLA + strain. ICAM-1 expression was increased with ATCC infection. Treatment of diabetic rats with a single dose of insulin restored CINC-1 levels in the PeLF for both strains; however, PMN migration, IL-4, and IFN-γ were restored in rats infected with the ATCC strain, whereas the PeLF concentrations of CINC-2, IL-1β, and IL-4 were increased in N315-infected animals. Insulin restored PMN migration and CINC-2 levels in the PeLF in ATCC-infected rats. After multiple treatments with insulin, the levels of IL-1β, IL-6, and IFN-γ were increased in the PeLF of diabetic rats after infection with either strain, and CINC-2 levels were restored in N315-infected animals. Conclusion: These results suggest that insulin distinctively modulates cytokine production or release, PMN leukocyte migration, and adhesion molecule expression during the course of peritonitis induced by different strains of S. aureus .

We sought to characterize the genetic context of bla OXA-72 gene in a carbapenem-resistant Acinetobacter pittii strain recovered from a hospitalized patient from Belém, North Brazil, in the Amazon region. We found that the bla OXA-72 gene was carried by a small plasmid, pIEC338SCox, that is 10,498 bp. The gene is flanked by XerC/XerD-like recombinase sites, which suggests that this gene was acquired onto this plasmid by recombination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aim: To perform a thorough characterization of the subgingival microbiota of shallow, moderate and deep sites in subjects with chronic periodontitis (ChP)., Material and Methods: Subgingival samples were collected from subjects with ChP (n = 3/category of probing depth: ≤3, 4-6 and ≥7 mm) and periodontal health (PH). Individual samples were submitted to 16S rDNA high- throughput sequencing and the analysis was made using mothur and R packages., Results: Nine subjects with ChP and seven with PH were included and 101 samples were evaluated. Thirteen phyla, 118 genera and 211 OTUs were detected. Taxa from Chloroflexi and Spirochaetes phyla were associated with initial stages of disease. Fretibacterium, Eubacterium[XI][G-6], Desulfobulbus, Peptostreptococcaceae[XI][G-1] and [G-3], Bacteroidetes[G-3], Bacteroidaceae[G-1] genera and Filifactor alocis, Fretibacterium fastidiosum, Johnsonella spHOT166, Peptostreptococcaceae[XIII][G-1]HOT113, Porphyromonas endodontalis and Treponema sp. HOT258, which are not conventionally associated with disease, increased with the deepening of the pockets and/or were elevated in ChP; while Streptococcus, Corynebacterium and Bergeyella genera were associated with PH (p < .05)., Conclusion: Striking differences were observed between the microbiota of shallow and moderate/deep sites, but not between moderate and deep sites in ChP subjects. Differences between shallow sites in PH and ChP were also observed. The characterized microbiota included known oral microorganisms and newly identified periodontal taxa, some of them not-yet-cultured., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We report changes in the molecular epidemiology of vanA-containing Enterococcus during the intra and interhospital spread of high-risk clones, in Southeastern Brazil. While VRE faecalis predominated during 1998 to 2006, a reversal has been observed in the last years, where VRE faecium belonging to ST114, ST203, ST412, ST478 and ST858 have become endemic., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

We report here the sequence of the entire chromosome of Staphylococcus aureus strain FCFHV36, a methicillin-resistant strain heterogeneously intermediate to vancomycin, bearing a type II staphylococcal chromosome cassette mec element (SCCmec), belonging to multilocus sequence type (MLST) 105, and isolated from a vertebra of a patient with osteomyelitis., (Copyright © 2015 McCulloch et al.)

We report the whole-genome sequence (WGS) of an in vitro susceptible derivative revertant mutant from a bloodstream isolate involved in a nosocomial outbreak in Brazil. The WGS comprises 2.5 Mb with 2,500 protein-coding sequences, 16rRNA genes, and 60 tRNA genes., (Copyright © 2015 de Almeida et al.)

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product., (Copyright © 2014 McCulloch et al.)

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

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.

Staphylococcus aureus is one of the most frequent mastitis causative agents in small ruminants. The expression of most virulence genes of S. aureus is controlled by an accessory gene regulator (agr) locus. This study aimed to ascertain the prevalence of the different agr groups and to evaluate the occurrence of encoding genes for cytotoxin, adhesins and toxins with superantigen activity in S. aureus isolates from milk of ewes with clinical and subclinical mastitis in sheep flocks raised for meat production The agr groups I and II were identified in both cases of clinical and subclinical mastitis. Neither the arg groups III and IV nor negative agr were found. The presence of cflA gene was identified in 100% of the isolates. The frequency of hla and lukE-D genes was high - 77.3 and 82.8%, respectively and all isolates from clinical mastitis presented these genes. The sec gene, either associated to tst gene or not, was identified only in isolates from subclinical mastitis. None of the following genes were identified: bbp, ebpS, cna, fnbB, icaA, icaD, bap, hlg, lukM-lukF-PV and se-a-b-d-e.

Staphylococcus aureus is one of the most important infectious mastitis causative agents in small ruminants. In order to know the distribution of Staph. aureus strains associated with infectious mastitis in flocks of sheep in the northeast of Brazil and establish whether these clones are related to the strains distributed internationally, this study analysed the genetic diversity of Staph. aureus isolates from cases of clinical and subclinical mastitis in ewes by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this research, 135 ewes with mastitis from 31 sheep flocks distributed in 15 districts were examined. Staph. aureus was isolated from sheep milk in 9 (29%) out of 31 herds located in 47% of the districts surveyed. MLST analysis allowed the identification of four STs (ST750, ST1728, ST1729 and ST1730). The last three with their respective novel alleles (glp-220; pta-182 and yqil-180) were recently reported in the Staph. aureus MLST database (http://www.mlst.net). Each novel allele showed only a nucleotide different from those already described. The occurrence of CC133 (ST750 and ST1729) in this study is in agreement with other reports that only a few clones of Staph. aureus seem to be responsible for most cases of mastitis in dairy farms and that some of these clones may have broad geographic distribution. However, the prevalence of CC5 (ST1728 and ST1730)--an important group related to cases of colonization or infection in humans--differs from previous studies by its widespread occurrence and may suggest human contamination followed by selective pressures of the allelic diversifications presented for these STs.

Background: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents., Methods: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (ζ) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations., Results: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 μg/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species., Conclusions: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.

Since Staphylococcus aureus can cause several types of diseases, the development of antibiotic resistance poses an even greater threat to public health. S. aureus is known to possess the adaptive capability to promptly respond to antibiotics, making it resistant and increasingly difficult to treat; methicillin-resistant strains of S. aureus are a major concern with regard to this species. Previous studies reported the identification of methicillin-resistant S. aureus in food, demonstrating that this can represent a source of S. aureus which may carry the mecA gene. Fifty-seven S. aureus isolates, previously obtained from different types of food, were screened by polymerase chain reaction with specific primers for the mecA gene, which mediates methicillin resistance. Five (9%) isolates showed the presence of mecA gene, demonstrating that food may contain microorganisms possessing resistance genes. This study emphasizes the need to include food as a possible source of S. aureus carrying mecA gene and the need to monitor these products. Moreover, this is the first report of the presence of mecA genes in S. aureus isolated from ready-to-eat food in Brazil and Latin America., (© Mary Ann Liebert, Inc.)

The aim of this study was to investigate the presence and prevalence of bla(TEM), bla(SHV), and bla(CTX-M) and bla(GES)-like genes, responsible for extended spectrum beta-lactamases (ESBLs) production in clinical isolates of Klebsiella pneumoniae collected from a Brazilian tertiary care hospital. Sixty-five ESBL producing K. pneumoniae isolates, collected between 2005 and 2007, were screened by polymerase chain reaction (PCR). Identification of bla genes was achieved by sequencing. Genotyping of ESBL producing K. pneumoniae was performed by the enterobacterial repetitive intergenic consensus-PCR with cluster analysis by the Dice coefficient. The presence of genes encoding ESBLs was confirmed in 59/65 (90.8%) isolates, comprising 20 bla(CTX-M-2), 14 bla(CTX-M-59), 12 bla(CTX-M-15), 9 bla(SHV-12), 1 bla(SHV-2), 1 bla(SHV-2a), 1 bla(SHV-5), and 1 bla(SHV-31) genes. The ESBL genes bla(SHV-12), bla(SHV-31), and bla(CTX-M-15), and the chromosome-encoded SHV-type beta-lactamase capable of hydrolyzing imipenem were detected in Brazil for the first time. The analysis of the enterobacterial repetitive intergenic consensus-PCR band patterns revealed a high rate of multiclonal bla(CTX-M) carrying K. pneumoniae isolates (70.8%), suggesting that dissemination of encoding plasmids is likely to be the major cause of the high prevalence of these genes among the K. pneumoniae isolates considered in this study., (© Mary Ann Liebert, Inc.)

Objective: The aim of this study was to characterize Staphylococcus aureus (MRSA) carriage in a dermatology unit., Methods: This was a prospective and descriptive study. Over the course of 26 weeks, surveillance cultures were collected weekly from the anterior nares and skin of all patients hospitalized in a 20-bed dermatology unit of a tertiary-care hospital. Samples from healthcare workers (HCWS) were cultured at the beginning and end of the study. Colonized patients were put under contact precautions, and basic infection control measures were enforced. Staphylococcus aureus colonization pressure was determined monthly. Colonized and non-colonized patients were compared, and isolates were evaluated for antimicrobial susceptibility, SCCmec type, virulence factors, and type., Results: Of the 142 patients evaluated, 64 (45%) were colonized by MRSA (39% hospital acquired; 25% community acquired; 36% indeterminate). Despite isolation precautions, hospital-acquired Staphylococcus aureus occurred in addition to the continuous entry of Staphylococcus aureus from the community. Colonization pressure increased from 13% to 59%, and pemphigus and other bullous diseases were associated with MRSA colonization. Eleven out of 71 HCWs (15%) were Staphylococcus aureus carriers, although only one worker carried a persistent clone. Of the hospital-acquired MRSA cases, 14/28 (50%) were SCCmec type IV (3 PFGE types), 13 were SCCmec type III (46%), and one had an indeterminate type. These types were also present among the community-acquired Staphylococcus aureus isolates. SSCmec type IV isolates were shown to be more susceptible than type III isolates. There were two cases of bloodstream infection, and the pvl and tst virulence genes were absent from all isolates., Conclusions: Dermatology patients were colonized by community- and hospital-acquired Staphylococcus aureus. Half of the nosocomial Staphylococcus aureus isolates were SCCmec type IV. Despite the identification of colonized patients and the subsequent contact precautions and room placement, Staphylococcus aureus colonization continued to occur, and colonization pressure increased. Pemphigus and other bullous diseases were associated with Staphylococcus aureus.

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculos isisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9%, 17.2% and 11.8% of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7% discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosis predominating.

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CMC, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta = 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of P. aeruginosa viability (10(7) CFU/mL) at 1 or 2 microg/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 microg/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.

It is known that Aeromonas spp. possess different chromosomal β-lactamase genes. Presence and phenotypic expression of bla TEM, bla SHV, and bla CTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of bla SHV and bla CTX-M genes was not observed, and bla TEM gene was verified in 91% of the isolates. Sequencing of 10 fragments showed the occurrence of bla TEM-116.

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.

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-beta-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-beta-lactamase and 16S rRNA methylase has a potential impact on the empirical management of complicated infections caused by P. aeruginosa.

Methicillin-resistant Staphylococcus aureus is an established nosocomial pathogen (HA-MRSA, hospital acquired MRSA), but has recently begun to appear in the community (CA-MRSA, community acquired MRSA). The cause of resistance to methicillin and all other beta-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII are distinguished. HA-MRSA disseminated worldwide and causes the majority of S. aureus nosocomial infections with a limited number of clones disseminated including the Brazilian Epidemic Clone (BEC, ST239-MRSA-III). CA-MRSA isolates are susceptible to non-beta-lactam antibiotics, usually isolated from healthy individuals which do not possess any unknown risk factors for MRSA infection and are associated with a larger clonal diversity compared with HA-MRSA. However, during recent years distinction between HA-MRSA and CA-MRSA is beginning to fade. Actually, knowledge about MRSA disseminating clones is required to implement any strategies to control the transmission of MRSA either within hospitals or in community. For this reason, rapid identification of strains is an important issue. The rate of HA-MRSA can be reduced substantially through the implementation of interventions strategies, even in settings where MRSA is endemic as in most Brazilian hospitals. However, these policies could be quite complicated in the light of an increasing CA-MRSA prevalence in healthcare facilities, considering that distinction between HA-MRSA and CA-MRSA has started to disappear.

Extended-spectrum beta-lactamases (ESBL) in enterobacteria are recognized worldwide as a great hospital problem. In this study, 127 ESBL-producing Enterobacteriaceae isolated in one year from inpatients and outpatients at a public teaching hospital at São Paulo, Brazil, were submitted to analysis by PCR with specific primers for bla SHV, bla TEM and bla CTX-M genes. From the 127 isolates, 96 (75.6%) Klebsiella pneumoniae, 12 (9.3%) Escherichia coli, 8 (6.2%) Morganella morganii, 3 (2.3%) Proteus mirabilis, 2 (1.6%) Klebsiella oxytoca, 2 (1.6%) Providencia rettgeri, 2 (1.6%) Providencia stuartti, 1 (0.8%) Enterobacter aerogenes and 1 (0.8%) Enterobacter cloacae were identified as ESBL producers. Bla SHV, bla TEM and bla CTX-M were detected in 63%, 17.3% and 33.9% strains, respectively. Pulsed field gel eletrophoresis genotyping of K. pneumoniae revealed four main molecular patterns and 29 unrelated profiles. PCR results showed a high variety of ESBL groups among strains, in nine different species. The results suggest the spread of resistance genes among genetically different strains of ESBL-producing K. pneumoniae in some hospital wards, and also that some strongly related strains were identified in different hospital wards, suggesting clonal spread in the institutional environment.

Aims: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil., Methods and Results: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively., Conclusions: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity., Significance and Impact of the Study: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.

Nineteen clonally related imipenem-resistant Acinetobacter baumannii isolates were recovered from eight intensive care unit patients. All isolates harboured bla OXA-51-like β-lactamase genes and showed the absence of 22 kDa fraction in outer membrane porin profile analysis. It suggests a combination of two mechanisms as responsible for carbapenem-resistant phenotypes.

Objective: To identify risk factors associated with nosocomial bloodstream infections caused by multiple clones of the staphylococcal cassette chromosome mec (SCCmec) type IV strain of methicillin-resistant Staphylococcus aureus (MRSA)., Design: An unmatched case-control study (at a ratio of 1:2) performed during the period from October 2002 through September 2003., Setting: A 2,000-bed tertiary care teaching hospital affiliated with the University of São Paulo in São Paulo, Brazil., Methods: Case patients (n=30) were defined either as patients who had a bloodstream infection due to SCCmec type IV MRSA diagnosed at least 48 hours after hospital admission or as neonates with the infection who were born in the hospital. Control patients (n=60) were defined as patients with SCCmec type III MRSA infection diagnosed at least 48 hours after hospital admission. Genes encoding virulence factors were studied in the isolates recovered from case patients, and molecular typing of the SCCmec type IV MRSA isolates was also done by pulsed-field gel electrophoresis and multilocus sequence typing., Results: In multivariate analysis, the following 3 variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: an age of less than 1 year, less frequent use of a central venous catheter (odds ratio [OR], 0.07 [95% confidence interval {CI}, 0.02-0.28]; p= .025), and female sex. A second analysis was performed that excluded the case and control patients from the neonatal unit, and, in multivariate analysis, the following variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: less frequent use of a central venous catheter (OR, 0.12 [95% CI, 0.03-0.55]; p= .007), lower Acute Physiology and Chronic Health Evaluation II score on admission (OR, 0.14 [95% CI, 0.03-0.61]; p= .009), less frequent surgery (OR, 0.21 [95% CI, 0.06-0.83]; p= .025), and female sex (OR, 5.70 [95% CI, 1.32-24.66]; p= .020). Of the 29 SCCmec type IV MRSA isolates recovered from case patients, none contained the Panton-Valentine leukocidin, gamma-hemolysin, enterotoxin B or C, or toxic shock syndrome toxin-1. All of the isolates contained genes for the LukE-LukD leukocidin and alpha-hemolysin. Genes for enterotoxin A were present in 1 isolate, and genes for beta-hemolysin were present in 3 isolates., Conclusions: "Classical" risk factors do not apply to patients infected with the SCCmec type IV strain of MRSA, which is an important cause of nosocomial bacteremia. This strain infects a patient population that is less ill and has had less frequent invasive procedures than a patient population infected with the multidrug-resistant strain of SCCmec type III MRSA. We found that virulence factors were rare and that Panton-Valentine leukocidin was absent. There were multiple clones of the SCCmec type IV strain in our hospital. Children under 1 year of age were at a higher risk. There was a predominant clone (sequence type 5) in this patient population.

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in São Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(IMP-1), aac(6')-31, and aadA1. Eight strains from the same hospital also carried another class 1 integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in São Paulo. These isolates appear to be the source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG), and within the inhA promoter and/or in structural gene. A small percentage (approximately 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh). Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Objectives: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined., Methods: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products., Results: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity., Conclusions: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Therapeutic activity of an effective and less nephrotoxic amphotericin B (AMB) formulation with dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (named DODAB/AMB) inspired this toxicity survey in mice. At low drug to lipid molar ratios, hepatotoxicity, spleen damage and blood changes in comparison to DOC/AMB (sodium desoxycholate/amphotericin B, Fungizone) are evaluated ultimately showing toxic effects associated to DODAB only. Swiss Webster female mice were given DODAB, DODAB/AMB or DOC/AMB intraperitonially (ip) for 10 consecutive days (0.4 mg/kg/day AMB; 80 mg/kg/day DODAB) and repeated dose-toxicity was evaluated at the end of the treatment period (on day 11) and after a recovery period of 6 months from biochemical and hematological parameters plus histopathological examination of spleen and liver. Both at day 11 and 180, DODAB in the formulation administered ip causes irreversible changes in spleen such as fibrosis and leukocytes infiltration as a consequence of the administration route. Whereas focal necrosis is induced by DODAB in liver at day 180, DOC/AMB causes more severe multifocal necrosis both at day 11 and day 180. In the kidneys, the novel formulation preserves integrity of tubules and glomeruli in contrast to the serious damage caused by DOC/AMB as shown previously. The majority of the toxic effects observed for the novel formulation were due to the DODAB carrier used at 10mg/mL, i.e., at a rather high concentration and further studies should minimize DODAB dose.