Vancomycin-resistant enterococci among haemodialysis patients in Portugal: Prevalence and molecular characterization of resistance,virulence and clonality

Introduction

Vancomycin-resistant enterococci (VRE) among haemodialysis patients has increased rapidly and, to date, there is no report of this incidence in Portugal.

Methods

A total of 121 faecal samples were collected from haemodialysis patients, and then tested for VRE. Antimicrobial resistance, virulence and multilocus sequence typing (MLST) were studied.

Results

VRE prevalence was 3.3%. Three VRE isolates, Enterococcus faecium, Enterococcus faecalis and Enterococcus raffinosus, were multi-resistant and vanA-positive. E. faecium and E. faecalis belonged to CC17 and CC2, respectively.

Conclusion

Haemodialysis patients in Portugal are colonized with virulent, multi-resistant enterococci from high-risk clonal complexes, representing a public health concern.     

Nuevo clon de Acinetobacter baumannii ST187 responsable de un brote en una unidad de cuidados intensivos

Introduction

An ICU-outbreak caused by a novel Acinetobacter baumannii clone is described.

Methods

An active search of carriers and environmental reservoirs was carried out. Carbapenemases genes were studied using multiplex-PCR and genotypic analysis by rep-PCR, PFGE and MLST.

Results

A total 26 infected patients and 10 carriers were identified. A. baumannii was recovered from infusion pumps, walls, floor and washbasins. Phenotypic/genotypic analysis showed clonal expansion of a unique clone ST-187 producer of type OXA-24 and OXA-51 carbapenemases.

Discussion

This is the first outbreak caused by ST-187 (ECI/GCI) multiresistant A. baumannii.     

Phenotypic and molecular characterization of serogroup C Neisseria meningitidis associated with an outbreak in Bahia,Brazil

Objective

To characterize meningococcal strains isolated from five cases of meningococcal disease (MD) associated with an outbreak in Trancoso – BA, occurred in October 2009. All cases, with the exception of a 39-year-old male, attended a dance party with approximately 1000 youngsters in a rural site.

Materials and methods

The epidemiological investigation was conducted by the Epidemiological Surveillance Service of Bahia State. Meningococcal strains were characterized at Adolfo Lutz Institute, the Brazilian National Reference Laboratory for Bacterial Meningitis by conventional techniques (serotype, serosubtype and antimicrobial susceptibility test) and by molecular methods (Pulsed-field gel electrophoresis – PFGE and Multilocus Sequence Typing – MLST).

Results

The PFGE showed 2 closely related restriction profiles, designated as PFGE types A and A1, having 92% relatedness to each other. MLST characterization showed both A and A1 clones were ST-3780, which belongs to the ST-103 complex. All isolates displayed the phenotype C:23:P1.5 and were susceptible to all antibiotics tested.

Conclusions

This is the first reported MD outbreak associated with serogroup C ST-103 complex in Brazil, as well as the party and illicit drug-use associated outbreak.     

Aplicación del análisis de los polimorfismos de los tamaños de los fragmentos de restricción del gen flaA amplificado por reacción en cadena de la polimerasa y resistotipo para identificar potenciales brotes de campilobacteriosis no diagnosticados en España

Introduction

Outbreaks of campylobacteriosis are infrequent and usually involve a low number of patients, although it is estimated that many more remain undiagnosed. The most successful techniques for outbreak investigation in Campylobacter spp. (PFGE, MLST) have the drawback of being laborious and not available in many laboratories.

Methods

During the year 2008, 352 isolates of C. jejuni and C. coli from 16 hospitals were received in our laboratory. All strains were genotyped by RFLP-PCR-flaA (flaA type) and phenotyped with their resistotype. It was established that the strains of the same species from the same hospital, isolated over a period of up to 11 days, with MIC values of ± 1 dilution with the same flaA type could belong to an outbreak. Strains that met these criteria would be later subtyped by KpnI-PFGE and MLST.

Results

A total of 23 out of 352 isolates, distributed in 10 groups, met the criteria for being associated with putative undiagnosed outbreaks. The similarity of the PFGE-profiles in 8 groups was greater than 95% among the isolates from each group. In 7 of the groups, the sequence types (MLST) were coincident.

Conclusions

The use of 2 easy markers (resistotype and RFLP-PCR-flaA) may detect isolates probably belonging to an undiagnosed outbreak of campylobacteriosis. Accurate diagnosis requires other molecular markers and epidemiological data of each isolate. The study suggests that, as in other countries, the number of outbreaks of campylobacteriosis in Spain is probably underestimated.     

Molecular epidemiology and antimicrobial susceptibility of Campylobacter coli clinical isolates

Introduction

Campylobacter spp. is a major cause of acute bacterial diarrhea in humans worldwide, and C. coli is responsible for 10% of the cases.

Materials and methods

A study was made of the antimicrobial susceptibility using the E-test^®, and the clonal relationship using PCR-RFLP, of the flaA gene, as well as PFGE techniques on 43 C. coli clinical isolates.

Results

Only 49% and 2% of the isolates were susceptible to erythromycin and ciprofloxacin, respectively. Imipenem and clindamyicn, with 100% and 84% of the strains, respectively, being susceptible, were the most active antimicrobials. The PCR-RFLP of flaA gene technique grouped fourteen isolates into six clusters, while the PFGE technique grouped eleven isolates into five clusters.

Conclusion

Ciprofloxacin and erythromycin are not suitable for the treatment of C. coli infections. Clindamycin could be considered as a therapeutic alternative in cases of enteritis, while imipenem is the best alternative for extra-intestinal infections. Both PFGE and PCR-RFLP can be useful to detect clones.     

Caracterización molecular de cepas de Staphylococcus aureus resistentes a meticilina ST398 en pacientes con infecciones cutáneas y sus familiares

Introduction

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of sequence type ST398 is a genetic lineage also described in human infections.

Methods

Cutaneous infections related with MRSA ST398 are described in 3 patients, two of them pig farmers. The MRSA nasal carriage by patients and their relatives was also studied. MRSA ST398 strains were typed (SCCmec, spa, agr and MLST) and the antimicrobial resistance pattern and virulence genes were determined.

Results

Twenty MRSA ST398 isolates were recovered in lesions of three patients, and in nasal samples of two patients and five relatives. Isolates were typed: spa-type t011 or t108; agr-type I and SCCmec IVa or V. MRSA strains were tetracycline-resistant and 15 of them showed a phenotype and genotype of multi-resistance, but they were free of tested virulence genes.

Conclusions

LA-MRSA ST398 is an emergent problem in our country, mainly associated with skin and soft tissue infections in people with professional relationships with pig farms. Tetracycline resistance is an important marker for MRSA ST398 detection.     

Staphylococcus coagulasa negativos resistentes al linezolid: características fenotípicas,genotípicas y sensibilidad a combinaciones de antibióticos

Objective

We recovered 22 coagulase-negative staphylococci isolates in our hospital to study their identity, susceptibility, epidemiological profile, linezolid resistance mechanisms, and the possibilities of different antibiotic combinations.

Methods

Isolate identification was performed using mass spectrometry (Vitek-MS, bioMérieux). Susceptibility testing was carried out with the Vitek-2 system and the broth microdilution method according to CLSI guidelines. Pulsed-field gel electrophoresis (PFGE) was performed to analyze the genetic relationship between isolates. Linezolid resistance mechanisms were evaluated by PCR/sequencing: presence of cfr gene, point mutations in domain V of 23S ribosomal RNA and additional ribosomal mutations (in the rplC, rplD and rplV genes).The in vitro activity of linezolid was investigated alone and in combination with another three antibiotics acting on different cellular targets, using E-test strips.

Results

Twenty isolates were identified as Staphylococcus epidermidis, and 2 as Staphylococcus hominis. PFGE showed that isolates belonged to diverse clones, 21 of them presented mutations in the domain V region of 23S rRNA and the cfr gene was found in 54.5%.Prior administration of linezolid was documented in most of cases.Linezolid in combination with gentamicin showed a synergistic activity in 45.5% of isolates.

Conclusions

Staphylococcus epidermidis was the most prevalent linezolid-resistant coagulase-negative staphylococci. All isolates showed increased MIC values compared to other anti-staphylococcal drugs and several linezolid resistance mechanisms. Our data suggest that linezolid plus gentamicin could be a synergistic combination against linezolid-resistant coagulase-negative staphylococci.     

Asociación en un mismo plásmido de blaVIM-1 y qnrS2 en Klebsiella pneumoniae y Klebsiella oxytoca aisladas en Sevilla

Background

Combined resistance to quinolones and β-lactams is common in Enterobacteriaceae. The appearance in enterobacteria coding for metallo-β-lactamases and determinants of plasmid-mediated quinolone resistance are an emerging problem in our country.

Methods

The susceptibility was determined by E-test. The resistance genes were detected by PCR and the corresponding plasmids were characterised.

Results

This study describes 2 strains (1 Klebsiella oxytoca, 1 Klebsiella pneumoniae) carrying the genes qnrS2 and blaVIM-1 in a transferable plasmid of 70-Kb isolated in surveillance cultures at the University Hospital Virgen Macarena in Seville.

Conclusion

This is the first combination of qnrS2 and bla_VIM-1 on the same non-typeable plasmid isolated in our centre.     

Escherichia coli O25b:H4/ST131 are prevalent in Spain and are often not associated with ESBL or quinolone resistance

Introduction

A multiresistant CTX-M-15-producing clonal group of Escherichia coli isolates, namely O25b:H4/ST131, has recently emerged in three continents. At this moment, appropriate studies to assess the real prevalence of this successful lineage are still scarce.

Methods

In a prospective study in the south of Spain, among all clinical E. coli isolates recovered in Seville during a 30 week period in 2010, ST131 was screened by using PCR for O25b/pabB3/B23 traits. ESBL enzymes were characterized by PCR and sequencing. Genetic relatedness was performed by XbaI PFGE.

Results

This clonal group was found to be prevalent (12.5% of all E. coli isolates), and only 37 (6.8% of ST131 isolates) were ESBL producers. Among 25 characterized ESBL-producing ST131 isolates, 96% harbored CTX-M-15. ST131 isolates were more frequently resistant to amoxicillin/clavulanate, aminoglycosides and fluoroquinolones in both ESBL and non-ESBL producers groups. XbaI PFGE performed on 88 ST131 isolates showed three pulsotypes, which included ≥4 isolates each (25% of all typed ST131 isolates), and 11 pulsotypes, which contained 2–3 isolates each. Three of 14 pulsotypes of this clonal group included both nalidixic acid-resistant and susceptible isolates, and five pulsotypes included both ESBL and non-ESBL producers.

Conclusions

Our findings suggest that O25b/ST131 is a prevalent clone in our area, and the observed prevalence of ESBL-producers within this clone is similar to that found in the total isolates of this species. Certain pulsotypes among ST131 clone that showed a greater expansion, and ESBL genes acquisition or quinolone resistance could explain part of this prevalence.     

Molecular epidemiology of fluoroquinolone resistance in invasive clinical isolates of Streptococcus pneumoniae in Seville

Introduction

Due to the emergence of drug-resistant pneumococcal isolates, new fluoroquinolones have been recommended for the treatment of pneumococcal infections. The purpose of this study was to establish surveillance, and to conduct molecular characterization, of fluoroquinolone-resistant Streptococcus pneumoniae in Seville.

Method

Norfloxacin-resistant S. pneumoniae isolates were characterized by quinolone resistance-determining region (QRDR) substitutions, reserpine-sensitive efflux, serotype and by pulsed-field gel electrophoresis (PFGE) patterns.

Results

Fourteen isolates (5.1%) showed an MIC > 16 μg/ml to norfloxacin. Eight of 10 adult isolates were susceptible to levofloxacin. The 4 infant isolates with norfloxacin MIC > 16 μg/ml were susceptible to levofloxacin. Seven of these 12 low-level-resistant isolates had mutations in ParC, while mutations both in ParC and GyrA genes were only detected in one of the two high-level-resistant isolates. All the isolates without QRDR substitutions that remained norfloxacin-resistant were positive for reserpine-inhibited efflux. The serotyping and PFGE revealed significant heterogeneity. We obtained 9 different profiles, 3 of which had two isolates each. Two of the isolates with the same pulsotype were from the same patient. The first isolate showed a mutation in the QRDR of ParC, and the second one had an additional GyrA mutation.

Conclusion

In our study a levofloxacin resistance rate of 0.7% was found among invasive isolates. Although resistance level is low, surveillance is necessary, especially to prevent cases of in vivo resistance development as reported.     

Caracterización de cepas de Staphylococcus epidermidis y S. haemolyticus resistentes a meticilina y linezolid en un hospital español

Introduction

Linezolid resistance is mainly due to mutations in the 23S rRNA target. The aim of this study was to characterize linezolid and methicillin resistant Staphylococcus epidermidis (SE-LM^R) and S. haemolyticus (SH-LM^R) strains detected in a Spanish hospital.

Methods

SE-LM^R and SH-LM^R strains obtained in the period June 2009-August 2011 in a second level hospital were recorded along with the epidemiological characteristics of the patients. These strains were typed, and their resistance, phenotype, genotype and the factors determining their virulence were analysed.

Results

Linezolid resistance was explained by the presence of G2603T mutation (23S rRNA) and aminoacid changes in L3 and L4 ribosomal proteins. The 25 SE-LM^R strains belonged to sequence type ST2, presented SCCmec type III, and two different PFGE patterns. The two SH-LM^R strains showed non-typeable SCCmec. SE-LM^R strains harboured the resistance genes aac(6’)-aph(2”), and dfrS1. SH-LM^R strains contained these genes and the gene erm(C). No lincomycin resistance mechanism was identified in SE-LM^R strains regardless of showing lincomycin resistance and diminished susceptibility to clindamycin.

Conclusions

Linezolid resistance is of concern in hospitals, and requires continued vigilance. Several linezolid resistance mechanisms (mutation in 23S RNAr and amino acid changes in L3 and L4) were identified in this study.     

Detección de la β-lactamasa de espectro extendido OXA-141 en cepas de Pseudomonas aeruginosa aisladas de pacientes con fibrosis quística

Introduction

The aims of this research were to study the presence of extended spectrum β-lactamases (ESBL) to investigate the location of the genes encoding these enzymes, and determine the clonal relationship of strains of ceftazidime-resistant Pseudomonas aeruginosa isolated from Mexican patients with cystic fibrosis.

Methods

We determined the resistance profile to 11 antibiotics (CLSI) and phenotypic ESBL detection following a disk diffusion method adapted for P. aeruginosa. Characterization of ESBL genes and integrons was performed by polymerase chain reaction (PCR) and DNA sequencing, while analysis of the clonal relationship was performed by pulsed field gel electrophoresis (PFGE).

Results

Of the 148 strains studied, 22 were resistant to ceftazidime, and analysis by PCR and sequencing revealed the presence of the gene bla_OXA-141 in 7 strains, 6 of which were resistant and one, susceptible to ceftazidime. In addition, bla_GES was detected in 11 strains. intI2 and intI3 genes were not detected by PCR, but in the 6 ceftazidime-resistant strains, the bla_OXA-141 gene was determined in a class 1 integron. Analysis of the clonal relationship of isolates showed that the majority of patients were infected during the study period with P. aeruginosa strains that exhibit different patterns, especially in individuals without a familial relationship.

Conclusions

This report demonstrates the existence of the bla_OXA-141 gene associated with a class 1 integron in several strains of P. aeruginosa, as well as bla_GES genes, and their location and variants are being studied by our research group. This, combined with the diversity of strains able to infect several susceptible individuals, suggests the risk of spread of P. aeruginosa-strain ESBL producers among Mexican populations with cystic fibrosis.     

A preliminary guideline for the assignment of methicillin-resistant Staphylococcus aureus to a Canadian pulsed-field gel electrophoresis epidemic type using spa typing

BACKGROUND

Increasing rates of methicillin-resistant Staphylococcus aureus (MRSA) infections on a global scale is a major health concern. In Canada, there are 10 known epidemic types of MRSA as determined by pulsed-field gel electrophoresis (PFGE). Despite the excellent discriminatory power of PFGE, there are several disadvantages of using this technique, such as high degree of labour intensity and the inability to easily develop an MRSA typing database due to the subjective interpretation of results.

OBJECTIVES

The purpose of the present study was to determine whether spa typing, an established DNA sequence-based typing method, could be used as an alternative to PFGE for the typing of Canadian MRSA (CMRSA) epidemic isolates.

RESULTS

spa types were determined for 1488 CMRSA isolates, and the method was analyzed for its ability to identify and cluster CMRSA1-10 strains. Minimal spanning tree analysis of 1452 spa types revealed individual clonal clusters for PFGE epidemic types CMRSA1, 2, 7 and 8, but spa typing could not distinguish CMRSA5 from CMRSA9 and CMRSA10, and CMRSA3 from CMRSA4 and CMRSA6. However, specific spa types were generally associated with only one PFGE epidemic type. Based on these results, a spa typing guideline for CMRSA isolates was developed and tested using the first 300 MRSA isolates received in 2007 through the Canadian Nosocomial Infection Surveillance Program.

CONCLUSIONS

The high concordance of spa types with PFGE epidemic types using this guideline demonstrated the feasibility of spa typing as a more rapid and less technically demanding alternative typing method for MRSA in Canada.     

Sensibilidad a azitromicina y otros antibióticos en aislados recientes de Salmonella,Shigella y Yersinia

Introduction

Azithromycin represents an alternative option to treat bacterial diarrhea when the antibiotic therapy is indicated. Little is known regarding the susceptibility to azithromycin in enteropathogens in Spain.

Methods

The MICs of azithromycin were determined by E-test against Salmonella non-typhi (SNT), Shigella and Yersinia isolates collected over the last three years (2010-2012). In addition, the susceptibility to other antibiotics usually used to treat gastrointestinal diseases was determined in these isolates by using a microdilution method.

Results

A total of 139 strains of SNT, Shigella and Yersinia were studied. All of them, except one strain, had a MIC ≤ 16 mg/L of azithromycin. In the adult population, 14.7% and 40.6% of SNT and Shigella isolates, respectively, were resistant to at least 2 of following antibiotics: amoxicillin, trimethoprim-sulfamethoxazole and ciprofloxacin. In the pediatric population, 10% of SNT clinical isolates and 28.6% (2/7) of Shigella isolates were resistant to amoxicillin and trimethoprim-sulfamethoxazole.

Conclusions

In our experience, azithromycin would be a useful antibiotic alternative to treat bacterial diarrhea.     

Caractéristiques clinicobiologiques des méningites à Enterovirus de l’adulte : étude de 59 cas

Purpose

To describe the epidemiological and clinical features of enteroviral meningitis as well as the biological profile of the cerebrospinal fluid (CSF).

Methods

A retrospective study conducted in a single centre between 2004 and 2008. All aseptic meningitis due to Enterovirus were included.

Results

Fifty-nine patients were included. The triad including fever, headache and neck stiffness was reported in 62% of patients. Twelve patients (20%) had a neutrophilic leukocytosis and 23 (39%) an elevated CRP level. Twenty-eight patients (47%) had a prominent neutrophilic reaction in the CSF and nine (15.2%) had a low glucose concentration. A presumptive anti-bacterial treatment was initiated in 47 patients (80%) for an average of 3.2 days.

Conclusion

Due to the lack of specificity of clinical features and biological manifestations of Enterovirus meningitis, the widespread use of real-time Enterovirus PCR is a priority for reducing the number of unnecessary anti-bacterial treatment. Guidelines based on clinical and biological features may be associated to help physicians in the differential diagnosis between bacterial and viral meningitides.     

Variantes pequeñas de Staphylococcus aureus: utilidad de distintas pruebas para su diagnóstico y estudio de sensibilidad

Introduction

Small colony variants of Staphylococcus aureus (SCVSA) are a sub-population with special features.

Methods

The phenotypic features and antibiotic susceptibility of four clinical isolates SCVSA were studied.

Results

Colonies grew in the usual culture media, except in Mueller Hinton. All isolates were resistant to ciprofloxacin and co-trimoxazole.

Discussion

As SCVSA are isolated with low frequency, it is necessary to determine the optimal methods for their identification and antibiotic susceptibility study.     

LCT-13910C > T polymorphism-associated lactose malabsorption and risk for colorectal cancer in Italy

Background

The activity of epithelial lactase (LCT) associates with a polymorphism 13910 bp upstream the LCT-encoding gene (LCT-13910C > T). The relationship between LCT-13910C > T polymorphism and risk for colorectal cancer is unclear.

Aims

We examined the relationship between the LCT-13910C > T polymorphism causing lactose intolerance and risk for colorectal cancer/polyps onset in the Italian population.

Patients and methods

793 subjects (306 with colorectal cancer, 176 with polyps and 311 controls) were genotyped for the LCT-13910C > T variant by TaqMan real time-PCR.

Results

Lactose malabsorption linked to the CC genotype did not associate with an increased risk for either colorectal cancer (OR = 1.041; 95% CI = 0.751–1.442; p = 0.868) or polyps (OR = 0.927; 95% CI = 0.630–1.363; p = 0.769). There was no association with colorectal cancer/polyps site. 60% of the subjects overall bore the CC genotype.

Conclusion

In the Italian population the LCT-13910C > T polymorphism is not associated to the risk for colorectal cancer or polyps.     

Resistencia a antibióticos y factores de virulencia en aislados clínicos de Salmonella enterica

Introduction

The increase of Salmonella enterica isolates multi-resistant to different antibiotics, including β-lactams and fluoroquinolones, is a problem of clinical importance. The dissemination of Salmonella Typhimurium resistant to ampicillin (AMP)-chloramphenicol (CHL)-streptomycin (STR)-sulphonamides and (SUL)-tetracycline (TET), that harbour the Salmonella Genomic Island type 1 (SGI1), and the acquisition of transferable genetic material have favoured the multi-resistance in this genus.

Methods

A total of 114 clinical S. enterica isolates were studied (period 2009-2010). The susceptibility to 20 antibiotics was determined by disc diffusion and microdilution. The antimicrobial resistance mechanisms and the integrons were analysed by PCR, and sequencing in the AMP^R isolates. In all the bla_PSE-1-positive isolates, the clonal relationship was determined by PFGE, as well as the presence of SGI1 and 29 virulence genes by PCR.

Results

Eighteen different serotypes were found among the 114 isolates studied, Typhimurium (61%) and Enteritidis (16%) being the most prevalent. High percentages of resistance to SUL (68%), TET (58%), AMP (55%) and STR (46%) were observed. The great majority (92%) of 63 AMP^R isolates were multi-resistant, with the AMP-STR-TET-SUL phenotype (19 isolates) being the most frequent one and associated with the bla_TEM-1b + strA-strB + tet(B) + sul2 genotype. Class 1 integrons (7 different structures) were observed in 48% AMP^R isolates, highlighting the bla_OXA-1 + aadA1 structure (8 isolates), one empty integron and non-classical integrons (5 isolates). The bla_PSE-1 gene was detected inside the classical SGI1 structure in 13 clonally-related isolates that showed the same virulence profile.

Conclusions

The high percentage of multi-resistant S. enterica isolates, especially associated to S. Typhimurium, to the AMP, STR, TET and SUL phenotype, and to the bla_TEM-1b + strA-strB + tet(B) + sul2 genotype, shows an important risk of possible failures in the treatment of serious infections caused by this serotype.