Evaluation of rep-PCR/DiversiLab versus PFGE and spa typing in genotyping methicillin-resistant Staphylococcus aureus (MRSA).

Pulsed-field gel electrophoresis (PFGE) is the 'gold standard' for genotyping of methicillin-resistant Staphylococcus aureus (MRSA); however, the DiversiLab (DL) system, based on rep-PCR, is faster, simpler and could be better adapted to daily routine hospital work. We genotyped 100 MRSA isolates using PFGE, DL, and spa typing, and evaluated the discriminatory power of each technique and the correlation between them by Simpson's index(SI) and adjusted Rand coefficient (ARI), respectively. The isolates were from clinical samples from eight hospitals in Extremadura (Spain) during 2010. DL separated the 100 MRSA into 18 patterns, with 69% of the isolates grouped into four predominant patterns. spa typing reported 17 spa types, classifying 69% of MRSA into two major types (t067 and t002). PFGE revealed the existence of 27 patterns, gathering 54% of MRSA into three pulse types (E8a, E7a and E7b). SI values were 0.819, 0.726, 0.887 and 0.460 for DL, spa typing, PFGE and CC-BURP, respectively. ARI values of DL over PFGE, spa typing and CC-BURP were 0.151, 0.321 and 0.071, respectively. DL has less discriminatory power than PFGE but more than spa typing. The concordance of DL with PFGE is low, primarily because DL does not discriminate between the three predominant MRSA pulse types in our environment.

Detection of new mutations conferring resistance to linezolid in glycopeptide-intermediate susceptibility Staphylococcus hominis subspecies hominis circulating in an intensive care unit.

Glycopeptides and linezolid are the most widely used antibiotics to treat infections by methicillin-resistant Staphylococcus spp. We report the presence of various isolates of methicillin-resistant S. hominis subsp. hominis with resistance to linezolid and reduced susceptibility to glycopeptides. We studied ten blood culture isolates of S. hominis subsp. hominis from nine patients admitted to our hospital. Etest was used to study susceptibility to antibiotics commonly prescribed against staphylococci. Domain V region of the 23S rRNA gene was amplified and sequenced to detect possible mutations that confer resistance to linezolid. Pulsed-field gel electrophoresis (PFGE) was used for the clonality study of isolates. All isolates were resistant to oxacillin, gentamicin, levofloxacin, cotrimoxazole, and linezolid, and susceptible to tigecycline and daptomycin. Nine of the isolates were resistant to erythromycin and clindamycin, and showed heterogeneous resistance to glycopeptides. C2190T, G2603T, and G2474T mutations were detected in domain V of the 23S rRNA gene. PFGE showed the presence of two different clones. This report alerts to the possible appearance of clinical strains of methicillin-resistant staphylococci with intermediate resistance to glycopeptides, resistance to linezolid, and multiple resistance to other second-line antibiotics.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Spain: a multicentre prevalence study (2002).

A point-prevalence study, performed in 2002 in 143 Spanish hospitals, collected 439 isolates of Staphylococcus aureus. Of these, 134 (30.5%) were resistant to methicillin (i.e., MRSA). Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were characterised by phage typing, pulsed-field gel electrophoresis (PFGE) after SmaI digestion, and SCCmec typing. The 134 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (88.8%), erythromycin (67.9%), clindamycin (59.7%), gentamicin (42.5%), mupirocin (17.9%), rifampicin (5.2%) and trimethoprim-sulphamethoxazole (5.2%). All of the isolates were susceptible to glycopeptides. Twenty-five resistance patterns were found, of which four accounted for 66% of the isolates. Phage group III was the most frequent (41.1%). PFGE revealed 31 different patterns, with ten major clones (including two predominant clones with variable antibiotypes that accounted for 43.3% of the MRSA isolates) and 21 sporadic patterns. Two isolates belonged to two variants of the Iberian clone (ST247-MRSA-I), one to the Brazilian clone (ST239-MRSA-III), and seven to the EMRSA-16 clone (ST36-MRSA-II). SCCmecIV accounted for 70.2% of the isolates (73.9% were type IVA), while SCCmecI, SCCmecII and SCCmecIII accounted for 22.1%, 6.9% and 0.8% of isolates, respectively, with three non-typeable isolates. Isolates of SCCmecIV and SCCmecIVA were predominantly nosocomial (95.8% and 97.1%, respectively). None of the isolates produced Panton-Valentine leukocidin. Thus, two clones carrying SCCmecIV and SCCmecIVA, respectively, were predominant among nosocomial MRSA isolates throughout Spain.

An analysis of the association between genotype and antimicrobial resistance in methicillin-resistant Staphylococcus aureus clinical isolates.

Genotyping methods are useful resources for the surveillance, detection, prevention and control of multidrug-resistant nosocomial agents, such as methicillin-resistant Staphylococcus aureus (MRSA). An understanding of the association between genotype and antibiotic susceptibility in MRSA clones may be useful in the surveillance of MRSA and to avoid inappropriate treatment future resistance. We genotyped MRSA clinical isolates from the Extremadura region of Spain using pulsed field electrophoresis (PFGE) and analyzed the spectrum of antibiotic susceptibility for each isolate to determine whether resistance is associated with specific genotypes. PFGE revealed six major genotypes: E8a (25%), E7b (17%), E7a (12%), E8B (8%), E10 (6%), and E20 (4%). Isolates with the genotypes E8a and E10 exhibit higher resistance ratios for levofloxacin than isolates with the other major pulsotypes. Similar results were obtained for isolates with the E20 pulsotype with respect to mupirocin. Although we identified no vancomycin-, tigecycline-, linezolid- or daptomycin-resistant strains, we observed significant differences in the mean MIC values obtained for some of these drugs among the major genotypes. Specifically, isolates with the E7b, E8b, and E20 genotypes have signif-icantly higher MICs of tigecycline, vancomycin and linezolid, respectively, than the most sensitive pulsotypes. Isolates with the E8b profile also exhibit a significantly higher rate of re-duced vancomycin susceptibility (RVS) (i.e., MIC between 1 and 2 mg/L) than clones with the E10 and E8a profiles. In conclusion, we report associations between genotype and antibiotic sensitivity that should be considered in programs for monitor-ing and controlling MRSA in health care settings.

In vivo attenuation and genetic evolution of a ST247-SCCmecI MRSA clone after 13 years of pathogenic bronchopulmonary colonization in a patient with cystic fibrosis: implications of the innate immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patient's innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

Biochemical, antimicrobial susceptibility and genotyping studies on Corynebacterium urealyticum isolates from diverse sources.

Thirty-two isolates of Corynebacterium urealyticum, isolated between 1991 and 1995, were studied by biochemical tests, phospholipid content, analysis of fatty and mycolic acids, ribotyping, whole-cell protein patterns and antimicrobial susceptibility to six antibiotics. Nineteen isolates were from human and human-related sources (HHRS); the remainder were from animal and animal-related sources (AARS). Most C. urealyticum isolates were similar in their biochemical and whole-cell protein profiles, although most HHRS isolates were alkaline phosphatase-positive (84%) and produced almost identical protein patterns, whereas AARS isolates were quite diverse. The qualitative composition of cellular fatty acids was identical for all isolates examined. Twelve different ribotypes were obtained with HindIII producing four-to-seven bands. Ribotypes 8, 9 and 10 were predominant in isolates from HHRS, whereas in isolates from AARS, ribotypes 5 and 6 predominated. AARS isolates were significantly less antibiotic-resistant, in comparison with HHRS isolates. Ribotyping appeared to be the most useful tool for strain characterisation.

Transmission of Streptococcus pyogenes causing successive infections in a family.

The objective of this study was to determine the characteristics of Streptococcus pyogenes isolated during a 10-month period from members of a family with infections and asymptomatic carriage. T-serotyping and pulsed-field gel electrophoresis confirmed that distinct GAS clones were introduced into the family over a short period of time.

Epidemiological typing of clinically significant strains of coagulase-negative staphylococci.

This study was undertaken to determine the characteristics of 202 isolates of coagulase-negative staphylococci (CNS) isolated from 78 patients (two-seven per patient) using biochemical identification, bacteriophage susceptibility patterns, reverse-typing, plasmid profiles, antimicrobial susceptibility testing and slime production. All the strains could be typed by using the six markers consecutively. In addition, we have been able to deduce the similarity of 58 strains from 24 patients by means of one marker or another (these are similar or different strains within one patient but not between one patient and another). The use of a combination of markers is the ideal method for typing the strains; thus making it possible to confirm if two or more isolates from the same patient are similar or not and if they produce infection or are simple contaminants.

[Pseudomonas aeruginosa: a multicenter study in 136 hospitals in Spain]. / Pseudomonas aeruginosa: estudio multicéntrico en 136 hospitales españoles.

A cooperative group of 136 Spanish hospitals identified 1014 isolates of P. aeruginosa in one week. It was estimated that Spanish microbiology laboratories identified 168 P. aeruginosa isolates per 100,000 inhabitants population and year (25 isolates for every 1,000 hospital admissions/year). P. aeruginosa was recovered in 5.3% of all the samples with bacterial isolates. Seventy-five percent of samples containing P. aeruginosa came from the lower respiratory tract, wound exudates, abscesses and urine. The three most common serotypes present in Spain were found to be 0:1, 0:4 and 0:11 and constituted more than 50% of all isolates. The antimicrobials active against more than 85% of all the isolates included: ceftazidime (85.2%), piperacillin-tazobactam (92.8%), imipenem (86.2%), meropenem (92.2%) amikacin (91.4%) and tobramycin (91.2%). The study showed a high rate of resistance to ciprofloxacin (22.7%) and gentamicin (31.1%). Of the 529 patients who underwent clinical follow-up, 25.5% showed P. aeruginosa colonization and the remaining 74.5% had clinical infections. We estimated an incidence rate of 88.4 patients infected with P. aeruginosa per 100,000 inhabitants and year (13.8 cases per 1000 hospital admissions and year). Overall, 42% were community acquired. The overall mortality in this study was 15%, and mortality attributable to P. aeruginosa infections was 5%. After logistical regression analysis, the two independent predictors of mortality were the presence of a rapidly fatal underlying condition and the presence of bacteremia. In Spain, P. aeruginosa is much more than a cause of severe nosocomial infections in immunocompromised patients.

[State of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) in Extremadura: susceptibility, clonality and role of community-associated MRSA]. / Situación actual de la infección por Staphylococcus aureus resistente a meticilina en Extremadura: sensibilidad, clonalidad, y protagonismo de la adquisición extrahospitalaria.

The correct surveillance and control of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) needs of update knowledge of its specific properties in each place. Our study aims to describe the current characteristics of infection due to MRSA in Extremadura. During 2010, 309 MRSA were collected from clinical samples in our region. A susceptibility test that included 17 antibiotics tested by AST -588 card Vitek 2 ® and E -test method was performed on all isolates. A sample of 100 strains, selected by stratified random sampling, were genotyped by pulsed field electrophoresis (PFGE). The prevalence of MRSA in Extremadura was 20.2%. Don Benito-Villanueva area showed the most prevalence and a higher incidence. Merida reported the most favourable situation, with a relatively low ratios of prevalence and incidence. The community acquired reached 44 % in the region, showing predominantly in less populated areas (Navalmoral and Coria). The most common multiresistant pattern was tobramycin-levofloxacin-erythromycin (44%), followed tobramycin-erythromycin-clindamycin (20%). No linezolid, daptomycin and tigecycline resistant strains were observed, but 42 % of the MRSA strains showed decreased susceptibility vancomycin (DSV). PFGE analysis reported 27 genotypes, with 3 major genotypes: E8a (25%), E7b (17%) and E7a (12%). The post-hoc statistical analysis did not reveal significant differences in the distribution of genotypes between different areas. However it revealed some trends that should be considered.

Molecular analysis of community-acquired methicillin-susceptible and resistant Staphylococcus aureus isolates recovered from bacteraemic and osteomyelitis infections in children from Tunisia.

Thirty-six children (27 boys, nine girls) that fulfilled CDC criteria for community-acquired infections were diagnosed with bacteraemia and/or osteomyelitis caused by Staphylococcus aureus during an 18-month period (2006-2008). Antibiotic susceptibility was determined by an agar dilution method. SCCmec type, carriage of pvl genes, agr type and spa-typing were determined using specific PCR protocols. Clonal relatedness was examined by pulsed field gel electrophoresis-SmaI and mutilocus sequence typing techniques. From the 36 isolates, eight (22%) corresponded to methicillin-resistant Staphylococcus aureus (MRSA) -t044/042-CC80/CC5-IVc-pvl(+) -agrIII/II. The highest genetic diversity was observed among the 28 community-acquired methicillin-susceptible S. aureus (CA-MSSA) isolates: 22 spa-variants that also grouped by multilocus sequence typing in CC1, CC5, CC6, CC8, CC30, CC80, CC97 and the singletons ST464, ST1467, ST1468 and ST1469. The pvl genes were detected in all eight CA-MRSA isolates and in eight CA-MSSA isolates (28%), being significantly more frequent among isolates causing osteoarticular infection (11 of 12, 92%) than in the bacteraemic isolates (six of 24, 25%). Based on patients' age, three groups were considered: newborns, infants and children. Bacteraemia was diagnosed in all newborns and infants, whereas in 42% of the children group osteomyelitis was the unique presentation. In most cases, the portal of entry was either the skin or unknown. In general, favourable outcome was observed, except in four cases-three of whom had severe complications and one died. In summary, we analysed the epidemiological and genetic background of community-acquired staphylococcal strains causing bacteraemic and/or osteomyelitis infections in children from Tunisia, describing three new sequence types and one novel spa type.

Trajectory and predictors of depression in a 12-month prospective study after the Madrid March 11 terrorist attacks.

BACKGROUND: Few longitudinal studies have examined the trajectory of and the risk factors for depression in a representative sample of the population exposed to terrorism. A 12 month prospective study was conducted among a sample of Madrid city residents after the March 11, 2004 terrorist attacks. We aimed to document the trajectories of depressive symptoms and determine the risk factors associated with these trajectories. METHODS: We conducted telephone surveys among a representative sample of Madrid citizens (N = 1589) to recruit baseline respondents approximately 1 month after the March 11 terrorist attacks. Participants were re-contacted at 6 and 12 months after baseline for further telephone interviews. RESULTS: Findings reveal heterogeneity in the longitudinal trajectories of depression ranging from the absence of depressive symptoms over time, to transient or chronic depression. Life and recent stressors, experiencing direct exposure to the attacks, personality traits, poor physical health and other psychological disorders were principally associated with a worse trajectory of depression after this event. CONCLUSIONS: Consistent with a stress diathesis model, ongoing stressors and intense event exposure are key drivers of a chronic depression trajectory after a mass traumatic event.

High frequency of Panton-Valentine leukocidin genes in invasive methicillin-susceptible Staphylococcus aureus strains and the relationship with methicillin-resistant Staphylococcus aureus in Córdoba, Argentina.

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Córdoba, Argentina, during 1999-2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Córdoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.

Prevalence and evolution of methicillin-resistant Staphylococcus aureus in Spanish hospitals between 1996 and 2002.

Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV.

Extracellular enzymes of fecal strains of Pseudomonas aeruginosa.

During a three year study 67 strains of Pseudomonas aeruginosa were isolated from stools of 67 patients with gastroenteritis. Serotypes 3 and 6 were the most frequent. The three strains that were serotype 11 were also the only beta-galactosidase producers. All strains were strongly pigmented. Over 90% of the isolates produced hemolysin, gelatinase, protease (caseinase), fibrinolysin, lecithinase and elastase. Fecal carriers of Pseudomonas aeruginosa can be considered a source of dissemination of potentially virulent and invasive strains.

Characterization of non-typable strains of Staphylococcus aureus from cases of hospital infection.

A high percentage of non-typable (NT) Staphylococcus aureus strains was isolated in Spanish hospitals during 1984 and 1985. Several alternative methods of typing were employed to study these isolates. These were: phage-typing at 1000 X RTD, phage-typing after heat-treatment (48 degrees C), thermal shock (56 degrees C), reverse-typing and induction of additional phages. Using these methods the number of NT isolates was reduced by 60%. Best results were obtained with heat-treatment. Additional phages and reverse-typing were also useful. A scheme for the study of outbreaks and sporadic cases caused by NT strains is proposed using the methods described.

Multicolonization of human nasopharynx due to Neisseria spp.

The colonization due to Neisseria spp. in the nasopharynx of forty healthy adults was studied by using a selective medium that allows the differentiation of Neisseria species and inhibits the rest of pharyngeal microbiota. The medium detected a variety of colonial morphology types and some metabolic characteristics of the isolates. We demonstrated the multicolonization by several Neisseria spp. in the same individual, and we isolated several strains of the same species, after analysis by pulsed-field gel electrophoresis (PFGE) patterns obtained from the different colonial types previously identified as the same species. The forty adults studied were colonized by 112 forms of Neisseria spp., and twelve colonization patterns were obtained: one species (45%), two (45%), three (7.5%) and four (2.5%). N. perflava-N. sicca, either alone or in combination with other species was the most frequent isolate (92.5%). The analysis of PFGE patterns obtained from different colonial types revealed the multicolonization by several strains of the same species in some individuals. This fact was found in N. perflava-N. sicca (50%) and N. mucosa (2.5%).

[Prevalence of serotype 0:12 among strains of P. aeruginosa causing nosocomial infection in Spain (1980-1991)]. / Prevalencia del serotipo 0:12 de cepas de P. aeruginosa productoras de infección nosocomial en España (1980-1991).

BACKGROUND: The aim of the present study was to characterize P. aeruginosa strains causing nosocomial infection in Spain between 1980-1990 with special emphasis on the incidence of serotype 0:12 strains. METHODS: 11,411 strains of P. aeruginosa from hospital-acquired infections were studied and epidemiologically characterized by phage-typing, serotyping and sensitivity to antimicrobial agents. The strains of the 0:12 serotype were analyzed by isoenzyme analysis. RESULTS: Although the major serotypes throughout the period studied were: 0:1, 0:6 and 0:11, the existence of serotype 0:12 strains (6%) were detected which had produced nosocomial outbreaks in surrounding countries. This serotype is homogeneous in that the epidemiologic markers and patterns of sensitivity to antibiotics and the multienzyme analysis demonstrate uniformity in the electrophoretic patterns of all the strains studied. CONCLUSIONS: The 0:12 serotype is in Spain indistinguishable by phage typing and studies of antibiotic sensitivity. It may be considered as being of clonal origin and is probably equal to that existing elsewhere in Europe.