Macrorestriction Analysis Of DNA Research Articles

MecA positive Staphylococcus spp. in bovine mastitis, milkers, milking environment, and the circulation of different MRSA clones at dairy cows farms in the Northeast region of Brazil

ABSTRACT: This study detected the presence and distribution of mecA in Staphylococcus spp. in the dairy production environment at farm level in Brazil. We analyzed 335 samples of mastitis cow milk, 15 samples of nostrils and hand swabs from milkers, 14 teat cup swabs, and 9 milking buckets swabs. Initially, the samples were subjected to microbiological analysis to detect Staphylococcus spp. and then S. aureus and mecA positive isolates were identified by PCR. All S. aureus isolates carrying the mecA genes were subjected to DNA macro-restriction analysis by Pulsed-Field Gel Electrophoresis (PFGE). The mecA gene was detected in 6/335 (1.78%) of mastitis cow milk, 5/15 (33.3%), and 5/15 (33.3%) of nostrils and hand swab, and 4/14 (28.5%) of the teat cup isolates. MRSA genotyping was performed by PFGE, a total of seven pulsotypes were grouped in two clusters. This study identified the occurrence and spread of MRSA at dairy environment of farms, and also the existence of distinct genetic profiles between isolates.

Genotypic and phenotypic heterogeneity in Streptococcus mutans isolated from diabetic patients in Rome, Italy.

Our study focuses on the antimicrobial susceptibility, genotypic and phenotypic heterogeneity, and serotype classification of the Streptococcus mutans isolated from type II diabetic patients (n = 25; age 42–68). Eighty-two percent of isolates were classified as serotype c. No serotype k was present. Macrorestriction analysis of genomic DNA of the isolates exhibited a clonal diversity that paralleled the phenotypic heterogeneity, which was also assessed in terms of biofilm forming ability. Isolates were susceptible to all the classes of antibiotics. In conclusion a great heterogeneity and no antimicrobial resistance were apparent in the considered S. mutans strains from diabetic patients.

Short communication: Streptococcus canis is able to establish a persistent udder infection in a dairy herd

Bovine mastitis caused by Streptococcus canis is relatively rare. Consequently, many epidemiologic aspects of the infection, including factors that mediate crossing of host species barriers by the pathogen, infectiousness of the microorganism to the mammary gland, and the course of the disease within a herd, are still not elucidated. Therefore, the aim of the present study was to describe results of a 15-mo observation of subclinical Strep. canis mastitis on a dairy farm housing 76 lactating Holstein-Friesian cows. Upon 3 visits to the farm during a period between April 2013 and June 2014, Strep. canis was cultured from milk samples of 17 (22.4% of the herd), 7 (9.6%), and 8 (11.3%) cows, respectively. The isolates obtained were characterized phenotypically by means of the API Strep identification kit (bioMérieux, Marcy l’Etoile, France), as well as genetically by using random amplified polymorphic DNA and macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis. All strains displayed the same biochemical features, and the molecular methods revealed that the isolates belonged to a single clone or were very closely related. Results of the study indicate that Strep. canis is capable of causing intramammary infections of long duration, behaving in a contagious manner. Because a persistently infected cow may serve as the source of Strep. canis infection for other animals, effective control of this type of udder infection within a herd may require similar measures to those adopted in Streptococcus agalactiae eradication programs.

The application of pulsed field gel electrophoresis for molecular typing of causative agents of especially dangerous infections

The macro-restriction analysis of the microorganism DNA with the use of gel electrophoresis in pulsed field (PFGE typing, pulse electrophoresis) is applied in molecular biology to study the clonal structure and typing of causative agents of infectious diseases. Determining the degree of the relationship and definition of epidemiological interrelations of studied isolates, as well as studying the evolutionary history of pathogens, is performed by comparing DNA restriction patterns. This review presents an analysis of the use of the pulse electrophoresis in molecular-epidemiological research and the study of phylogeny of especially dangerous infections, cholera, and plague. The possibility of genetic heterogeneity of the Vibrio cholerae and Yersinia pestis populations is demonstrated; territorial and epidemiological characteristics of the spread of different isolate pulso-types, problems and prospects of the PFGE typing method in the system of epidemiological surveillance of cholera and plague in Russia are discussed.

Differentiation of strains from the Bacillus cereus group by RFLP‐PFGE genomic fingerprinting

Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B.cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food-borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A-E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP-PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products.

Single-Nucleotide Polymorphism Genotyping Identifies a Locally Endemic Clone of Methicillin-Resistant Staphylococcus aureus

We developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed ‘t003-X’) within the radiation of methicillin-resistant Staphylococcus aureus (MRSA) ST225. The TaqMan assay achieved 98% typeability and results were fully concordant with DNA sequencing. By applying this assay to 305 ST225 isolates from an international collection, we demonstrate that clade t003-X is endemic in a single acute-care hospital in Germany at least since 2006, where it has caused a substantial proportion of infections. The strain was also detected in another hospital located 16 kilometers away. Strikingly, however, clade t003-X was not found in 62 other hospitals throughout Germany nor among isolates from other countries, and, hence, displayed a very restricted geographical distribution. Consequently, our results show that SNP-typing may be useful to identify and track MRSA clones that are specific to individual healthcare institutions. In contrast, the spatial dissemination pattern observed here had not been resolved by other typing procedures, including multilocus sequence typing (MLST), spa typing, DNA macrorestriction, and multilocus variable-number tandem repeat analysis (MLVA).

Molecular Typing in Public Health Laboratories: From an Academic Indulgence to an Infection Control Imperative

Using three Austrian case studies, the variegated applications of molecular typing in today's public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.

Change in Antimicrobial Resistance Pattern in Salmonella enterica serovar Typhimurium Isolates Detected in a Beef Cattle Farm

Multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates with four different antimicrobial resistance patterns obtained from a beef cattle farm were characterized to determine their clonality. Macrorestriction analysis of genomic DNA revealed that these four isolates are closely related to each other and can be classified as a newly emerged pulsed-field gel electrophoresis type among cattle: cluster VII. Three of the four isolates showed resistance to extended-spectrum cephalosporins (ESCs), and this resistance was mediated by AmpC β-lactamase encoded by the bla(CMY-2) gene in a 190-kbp IncA/C plasmid. Results of restriction analysis and IncA/C backbone PCR suggest that the three 190-kbp plasmids are identical and that a 70-kbp IncA/C plasmid of the ESC-susceptible isolate is derived from the 190-kbp plasmid by a deletion event. Three isolates harboured a virulence-resistance plasmid (165 or 180 kbp), and restriction analysis revealed that these plasmids were identical or closely related to each other. These results suggest that the four S. Typhimurium cluster VII isolates originate from a common ancestor that probably invaded the farm prior to the salmonellosis outbreak. Antimicrobial resistance patterns may not necessarily reflect the relationships of the isolates.

Toxigenic status of Staphylococcus aureus isolated from bovine raw milk and Minas frescal cheese in Brazil.

A group of 291 Staphylococcus aureus isolates from mastitic cow's milk (n = 125), bulk tank milk (n = 96), and Minas frescal cheese (n = 70) were screened for staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, selj, and sell) and for the tst-1 gene encoding staphylococcal toxic shock syndrome toxin 1 by PCR assay. A total of 109 (37.5%) of the isolates were positive for at least one of these 11 genes, and 23 distinct genotypes of toxin genes were observed. Of the S. aureus isolates bearing SE genes, 17 (13.6%) were from mastitic cow's milk, 41 (41.7%) were from bulk tank milk, and 51 (72.9%) were from Minas frescal cheese. The occurrence of exclusively more recently described SE genes (seg through sell) was considerably higher (87 of 109 PCR-positive strains) than that of classical SE genes (sea through see, 15 strains). The SE genes most commonly detected were seg and sei; they were found alone or in different combinations with other toxin genes, but in 60.8% of the cases they were codetected. No strain possessed see. The tst-1 gene was found in eight isolates but none from mastitic cow's milk. Macrorestriction analysis of chromosomal DNA from 89 S. aureus isolates positive for SE gene(s) was conducted with the enzyme SmaI. Fifty-five distinct pulsed-field gel electrophoresis patterns were found, demonstrating a lack of predominance of any specific clone. A second enzyme, Apa I, used for some isolates was less discriminating than Sma I. The high genotype diversity of potential toxigenic S. aureus strains found in this study, especially from Minas frescal cheese, suggests various sources of contamination. Efforts from the entire production chain are required to improve consumer safety.

Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions

The whole cell protein and macrorestriction analysis of DNA of Enterobacteriaceae isolates recovered from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO 2–30% O 2–30% N 2 in the presence (MAP+) and absence (MAP−) of oregano essential oil were studied. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) profiles obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups. Moreover, application of a modified PFGE protocol with XbaI restriction, resulted into 19 different fingerprints. The Enterobacteriaceae community of fresh meat consisted of Serratia liquefaciens and Serratia proteamaculans. S. liquefaciens strain VK23 was the dominant isolate of Enterobacteriaceae for the most conditions adopted, except 10 °C and 15 °C under MAP + and 10 °C under MAP−. In the latter cases, Hafnia alvei represented the dominant fingerprint. Citrobacter freundii was recovered from minced beef stored aerobically, while H. alvei and Proteus vulgaris were recovered under MAP. Storage conditions affected the Enterobacteriaceae community; modified atmosphere packaging increased both species and strain diversity.

Multidrug resistant and sensitive strains of Pseudomonas aeruginosa: Establishing clonal relationship by Pulsed Field Gel Electrophoresis and in vitro antibiotic synergy testing by E test

Background: The emergence of multidrug resistant Pseudomonas aeruginosa (MDRPA) as one of the leading nosocomial pathogens imposes a serious health threat due to limited antibiotic options. In this study, a sample of clonally distinct MDRPA and sensitive strains of Pseudomonas aeruginosa (SSPA) were tested against various antibiotics combinations to establish synergistic activity against these strains. Methods: A total of 39 retrospectively collected MDRPA strains isolated over three years with a particular antibiogram (sensitive to colistin but resistant to ceftazidime, cefepime, cefoperazone, cefoperazone/sulbactam , ciprofloxacin, imipenem, meropenem, piperacillin, piperacillintazobactam, gentamicin, netilmicin, with varying susceptibility to amikacin) and 19 SSPA collected prospectively were genotyped using Spe1 DNA macrorestriction analysis and pulsedfield gel electrophoresis (PFGE). The strains were from different patients from various sites (blood, tissue, fluid, catheter tip, bronchiol alveolar lavage (BAL), urine, sputum, swab, tracheal secretion and nasal swab). Clonal relatedness was determined at �85% level of similarity and grouped into different clusters. A representative strain from each cluster was tested against 10 different paired combinations of various antimicrobials (�-lactams, quinolone, aminoglycoside, tigecycline, rifampicin and colistin) to establish synergistic activity against these strains using the investigational E test fixed ratio method. Results: There were 46 distinct clones out of a total of 58 MDRPA and SSPA resolved by PFGE from which 10 clonally distinct strains (8 MDRPA and 2 SSPA strains) were selected for synergy testing. The antibiotic combination which most frequently demonstrated synergy was cefepime with amikacin (4/10 strains). Other synergistic combinations were aztreonam with either ceftazidime or cefepime, and meropenem with ciprofloxacin (3/10 strains respectively), ceftazidime with either ciprofloxacin or amikacin (2/10 strains respectively) and imipenem with ciprofloxacin (1/10 strains). The combination of ceftazidime with amikacin was antagonistic in 1/10 strains. The other antibiotics combinations were either additive or indifferent. Conclusion: PFGE was a useful and discriminative molecular subtyping tool to establish clonal relatedness among strains. Results of antimicrobial synergy testing indicate that although certain combinations may act synergistically, it was still strain dependent. These preliminary findings require further confirmation with testing of a larger number of isolates.

PCR melting profile (PCR MP) - a new tool for differentiation of Candida albicansstrains

BackgroundWe have previously reported the use of PCR Melting Profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains.MethodsIn total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland) were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE) and RAPD techniques.ResultsThe genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD.ConclusionData presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

Typing of Campylobacter jejuni Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.

Analysis of Slovenian MRSA strains with susceptibility patterns suggestive of CA-MRSA

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) differs from healthcare-associated MRSA (HA-MRSA) in its molecular and microbiological characteristics. Six Slovenian regional public health institutes and the National Institute of Public Health took part in monitoring CA-MRSA infections. S. aureus isolates resistant to oxacillin and susceptible to > or = two of the four antibiotics ciprofloxacin, erythromycin, clindamycin or gentamicin were defined as CA-MRSA and further analyzed. The presence of the gene for Panton-Valentine leukocidin (PVL) was confirmed using PCR, the type of staphylococcal cassette chromosome (SCCmec) using multiplex PCR, and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE). A total of 31 strains from 31 patients were analyzed during a period of 21 months: 23 specimens were sent from hospitals, six from primary care, two from a long-term care facility. All 31 isolates contained the gene mecA. Sixteen (51.6%) isolates were identified as SCCmec type IV, three isolates were PVL positive. Using PFGE, the CA-MRSA strains were classified into 15 similarity groups. Results of antibiotic susceptibility showed there were five resistance types among the 31 strains. Simultaneous resistance against ciprofloxacin and gentamicin was often associated with the presence of SCCmec type I, strongly resembling HA-MRSA. PVL-positive strains of CA-MRSA have been isolated in Slovenia only rarely. We will continue to monitor strains of MRSA in order to obtain the complete microbiological and epidemiological features.

Molecular Characterization ofStenotrophomonas maltophiliaIsolates from Cystic Fibrosis Patients and the Hospital Environment

To ascertain whether cystic fibrosis (CF) patients are colonized or infected with unique or multiple strains of Stenotrophomonas maltophilia; to understand whether some strains colonize or infect more than 1 patient, indicating clonal spread; and to explore the molecular heterogeneity of hospital water isolates and their correlation with clinical isolates. The regional CF center of Policlinico "Umberto I" of Rome, Italy. The study was carried out on a random sample of S. maltophilia isolates (n = 110) collected from CF patients (n = 50) during the period 2002-2005 and on 24 water isolates obtained during a monitoring program in the first 6 months of 2005. Home environmental samplings were not performed. All isolates, which were recovered from cultures of specimens obtained in both inpatient and outpatient settings, were genotyped with DNA macrorestriction analysis with the restriction enzyme XbaI and pulsed-field gel electrophoresis. One-third of the patients with repeated episodes of S. maltophilia infection or colonization hosted more than 1 strain. A potential transmission, defined as the isolation of the same strain in 2 or more patients, occurred 5 times, showing a frequency of potential transmission episodes slightly higher than previously reported. Water, taps, and sinks of the different rooms of the CF center tended to be persistently colonized with the same strain of S. maltophilia, with no correlation between clinical and water-associated isolates. The study does not provide sufficient data to conclude definitively that isolation of colonized or infected CF patients and control of hospital water systems contamination would be beneficial infection control measures. Epidemiologic analytical studies that correlate the presence of S. maltophilia with clinical outcomes are strongly needed.

Clustering and Risk Factors of Methicillin-Resistant Staphylococcus aureus Carriage in Two Italian Long-Term Care Facilities

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized agent of health care-associated infections in long-term care facilities, but few data about the circulation of MRSA in this setting in Italy are available. The aim of the study is to determine the prevalence and risk factors for MRSA carriage in nursing home residents in Vicenza (northeastern Italy). A point prevalence survey was conducted in two long-term care facilities (subdivided into 15 wards) from 12 June 2006 to 6 July 2006. Anterior nasal swabs were obtained from residents and laboratory screening for MRSA was performed; full antibiotic susceptibility was assessed in MRSA isolates. Macrorestriction analysis of chromosomal DNA was carried out by pulsed field gel electrophoresis (PFGE). For each subject, demographic data, length of stay, dependency, cognitive function, presence of medical devices, comorbidities, current and previous antibiotic treatment, previous hospital admission and presence of infection were assessed on the day of sample collection. Factors that were found to be significantly associated with MRSA carriage at univariate analysis were introduced into multilevel logistic regression models in order to estimate the odds ratios (OR) with 95% confidence intervals (CI) for the risk of MRSA colonization, taking into account the clustering of patients within wards. Nasal swabs were obtained in 551 subjects; overall 43 MRSA carriers were detected (7.8%; CI = 5.7-10.4%). The rate of nasal carriers was very similar in the two institutions, and varied from 0% (0/36) to 18% (7/39) between wards. Only two out of 15 wards were found to have no MRSA carriers; overall, three pairs of colonized roommates were detected. Upon multilevel logistic regression, the risk of MRSA carriage was increased in patients with cancer (OR = 6.4; CI = 2.5-16.4), in those that had undergone recent hospitalization (OR = 2.2; CI = 1.0-4.4), and it reached OR = 4.0 (CI = 1.7-9.9) in those with three or more antibiotic treatments in the previous year; about 10% of the variability in MRSA carriage could be attributed to differences between wards. Pulsed field gel electrophoresis analysis permitted the definition of six clusters; two of these comprised 78.6% of the studied isolates and were quite similar, with one being more strongly represented among subjects hospitalized in the previous 12 months. All of the MRSA strains were resistant to ciprofloxacine; nevertheless, the majority were susceptible to most other non-betalactam antibiotics. The study suggests that nursing homes are a significant reservoir for MRSA. Statistical and PFGE analyses indicate a scenario where MRSA seems to be endemic and individual risk factors, namely recent hospitalizations and repeated antibiotic treatments, play a major role in the selection of drug-resistant organisms. Infection control measures should be coordinated among different health care settings, and the appropriate use of antibiotics has emerged as an important issue for improving the quality of care.

Efficient removal of attached biofilm in a naturally contaminated colonoscope using detachment-promoting agents

We would like to report a successful ‘last chance’ operation during which a highly contaminated colonoscope was effectively treated using an anti-biofilm procedure based on a synergistic combination of detachment-promoting agents (DPAs). Various authors have described the considerable difficulties presented by the removal of attached bacteria from endoscope channels. 1 Spach D.H. Silverstein F.E. Stamm W.E. Transmission of infection by gastrointestinal endoscopy and bronchoscopy. Ann Intern Med. 1993; 118: 117-128 Crossref PubMed Scopus (437) Google Scholar , 2 Struelens M.J. Rost F. Deplano A. et al. Pseudomonas aeroginosa and Enterobacteriaceae bacteremia after biliary endoscopy: an outbreak investigation using DNA macrorestriction analysis. Am J Med. 1993; 95: 489-498 Abstract Full Text PDF PubMed Scopus (136) Google Scholar , 3 Birnie G.G. Quigley E.M. Clements G.B. Follet E.A. Watkinson G. Endoscopic transmission of hepatitis B virus. Gut. 1983; 24: 171-174 Crossref PubMed Scopus (183) Google Scholar We agree with Vickery et al. who considered that the persistence of this attached biomass on endoscope channels is often not due to inadequate compliance with reprocessing protocols, but usually caused by the low efficiency of current reprocessing protocols that do not totally eliminate attached micro-organisms. 4 Vickery K. Pajkos A. Cossart Y. Removal of biofilms from endoscopes: evaluation of detergent efficiency. Am J Infect Control. 2004; 32: 170-176 Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar We previously demonstrated that the use of a cleaning agent according to the Pronetron® procedure, prior to disinfection in compliance with French guidelines, totally detached a preformed biofilm from the inner surface of an operating channel. 5 Anonymous. Circulaire DHOS/E2/DGS/SD5C/2003/N°591 relative aux modalités de traitement manuel pour la désinfection des endoscopes non autoclavables dans les lieux de soins. Ministère de la santé, de la famille et des personnes handicapées. Paris: 2003. Google Scholar , 6 Marion K. Freney J. James G. Bergeron E. Renaud F.N.R. Costerton J.W. Using an efficient biofilm detaching agent: an essential step for the improvement of endoscope reprocessing protocols. J Hosp Infect. 2006; 64: 136-142 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar This corrective procedure consists of the sequential action of two DPA-based products: a multi-enzymatic solution (Pronetron α) that digests the anchorage points of the micro-organisms to the support, and an enriched detergent solution (Pronetron β Plus) that detaches the whole biomass from the support.

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be “the gold standard” for epidemiological studies.

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes ( sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene ( tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0 × 10 3 CFU/ml. Farm size had no significant effect ( P > 0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes ( seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.

Molecular Epidemiology of Imipenem-Resistant Acinetobacter haemolyticus and Acinetobacter baumannii Isolates Carrying Plasmid-Mediated OXA-40 from a Portuguese Hospital

Major outbreaks of multidrug-resistant Acinetobacter baumannii associated with nosocomial infections have been increasingly reported worldwide (1, 10, 12). The endemicity of an OXA-24/40-producing A. baumannii clone associated with mortality events in Portugal has been observed at numerous hospitals within the Iberian Peninsula (5, 6, 10). Inversely, Acinetobacter haemolyticus, isolated only occasionally from clinical samples (9), usually presents susceptibility to different antibiotics, including β-lactams (13). The isolation of two carbapenem-resistant A. haemolyticus strains prompted us to assess the relative contribution of clonal spread to the observed high rate of carbapenem-resistant Acinetobacter spp. in a general hospital in Porto, Portugal. Between January 2001 and October 2004, 224 imipenem-resistant Acinetobacter spp. were collected from several specimen sources and different hospital wards, where A. baumannii was associated with nosocomial infections and colonizations for several months (Table ​(Table1).1). Imipenem resistance significantly increased from 2001 to 2002 and from 2002 to 2003. Macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis (5) and 16S rRNA gene sequencing, performed for each clone and species representative, showed that, with the exception of two clonally related A. haemolyticus isolates, the remainder were A. baumannii isolates, distributed among three different pulsotypes. Clonal dissemination of two major pulsotypes (A and B), widespread throughout the hospital, contributed to the observed A. baumannii imipenem resistance, which has persisted since at least 2001 despite several elimination attempts, including the use of polymyxin. Pulsotype B was predominant from 2001 to 2002, after which clone A emerged as the dominant type (Table ​(Table1).1). This clone was found to be identical to the previously described Iberian OXA-24/40-producing clone (5). Pulsotype C, with only two isolates, seemed to represent a sporadic event within the observed prevalence of clones A and B. Antimicrobial susceptibilities varied among isolates according to clones (Table ​(Table2).2). A. haemolyticus isolates presented resistance to all β-lactams, with the exception of cefepime, ceftazidime, and aztreonam. All Acinetobacter sp. isolates were resistant to ciprofloxacin, whereas susceptibility to aminoglycosides was variable. Only 11 isolates (including the two A. haemolyticus isolates) showed a colistin MIC of ≥4 μg/ml (2). However, when the recently updated CLSI susceptible interpretative criterion of ≤2 μg/ml (3, 8) was applied, the susceptibility rate dropped from 96.1% to 92.1%. Detection of carbapenemase production, ulteriorly identified as an OXA-24/40 enzyme, was performed as previously described (5) and was positive only for clone A A. baumannii isolates and, for the first time, A. haemolyticus isolates. Hybridization assays after both S1 nuclease digestion and I-CeuI digestion, performed as previously described (7), revealed that although some clone A A. baumannii isolates showed a chromosome-positive signal (ca. 150 kb) for the blaOXA-24/40 probe, most also presented a positive hybridization in plasmidic bands of ca. 180 kb and ca. 30 kb. Similar hybridization signals were observed in the A. haemolyticus isolates. Further studies on plasmid characterization, assessing the homology among different plasmids, are ongoing. TABLE 1. Clinical data for imipenem-resistant Acinetobacter spp.a TABLE 2. In vitro susceptibilities of imipenem-resistant Acinetobacter sp. clinical isolates We describe, for the first time, the presence of an OXA-24/40 enzyme in an A. haemolyticus clinical isolate. Although the spread of OXA-24/40, both in the Iberian Peninsula and in France, has been correlated with the progressive dissemination of a single A. baumannii clone, the observation of this enzyme in a different, previously unreported, genomic species, A. haemolyticus, poses new questions on OXA-24/40 dissemination. It now seems reasonable to suspect a horizontal dissemination of the blaOXA-40 gene between different species, an ability supported by the observation of this enzyme, previously described as chromosomally encoded (7), in a plasmid. Notwithstanding, the dissemination of “successful” clones may possibly contribute to the high rates and persistence of imipenem-resistant A. baumannii isolates (4).