Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.     

Nontypeable bacteriophage patterns of methicillin-resistant Staphylococcus aureus involved in a hospital outbreak.

Of 93 strains of Staphylococcus aureus isolated from inpatient wards of Ismailia General Hospital, 48 (51%) were proven to be methicillin resistant (MR). Of these MR S. aureus strains, 44 were isolated from patients and 4 were isolated from healthy carriers, who were newly arrived interns working in the same wards. Bacteriophage patterns of MR S. aureus were identified by using routine test dilution (RTD) and 100-fold dilutions (100 RTD) of phages. Of these 48 strains, 37 (75%) (33 from patients and 4 from interns) were nontypeable when using RTD and 100 RTD of phages. Of the other 11 strains, 8 were nontypeable by RTD of phages, but 5 of them had the phage pattern D11/1136 when tested by 100 RTD. Three strains had the phage pattern 3A/3C/55/71, and three strains had different phage patterns, 29/81, 96, and 95/D11. The finding of colonization with virulent MR S. aureus strains in interns working on the wards in which these patients were located suggested that new strategies for control of MR S. aureus nosocomial infections must be considered and evaluated.     

Epidemiological typing of methicillin-resistant Staphylococcus aureus outbreak isolates by pulsed-field gel electrophoresis and antibiogram

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens. In April 1997, there were five MRSA-infected patients among 16 patients in the Neonatal Intensive Care Unit (NICU), Seoul National University Hospital, which is a tertiary-care hospital with 1,500 beds. The infections had spread from twin patients with MRSA who had transferred from Hospital C. MRSA was isolated from the axilla of 15 (94%) of the 16 patients, including the two patients with obvious infections. Three (19%) of 16 doctors and nine (30%) of 30 nurses had MRSA colonization of the anterior nares. Six different PFGE patterns (A through F) were identified in the 53 isolates of MRSA tested. Twelve of 13 isolates from infected sites of five patients showed pattern F. Three MRSA strains obtained from hospital C showed closely or possibly related pattern F. MRSA of type F was isolated from three of 16 patients' axilla, and one of 3 doctors' and three of 30 nurses' nasal swabs. The antibiogram code for 12 of 13 MRSA isolates from five infected patients was 66,754. PFGE patterns of these isolates were either F, F1, F2 or Fa. Only one of three strains isolated from clinical specimens of patients in Hospital C showed the antibiogram code 66754, although they were all PFGE types F1 and Fa. In conclusion, the presumptive sources of the outbreak of MRSA infection in NICU were the twin patients transferred from hospital C. Antibiogram correlated reasonably well to the PFGE type. An effective notification system is needed when a MRSA-infected patient is transferred to another hospital to control the spread of the infection.     

Resistance to desiccation and skin fatty acids in outbreak strains of methicillin-resistant Staphylococcus aureus.

Resistance to desiccation and to skin fatty acids was measured in three groups of methicillin-resistant Staphylococcus aureus (MRSA) strains and a group of control strains. Organisms from a large outbreak on a special care baby unit (SCBU), where MRSA had been isolated from staff hands but not from the environment, were significantly more sensitive to drying than strains from a burns unit where extensive environmental contamination had been demonstrated. MRSA from other wards, in the same hospital but not associated with large outbreaks, gave heterogeneous results. Fatty-acid resistance, determined by an agar dilution method, was not associated with strain origin. Some epidemic strains of MRSA were relatively sensitive to desiccation, and the abilities of such strains to spread widely on a SCBU by the hand-borne route could not be explained by enhanced resistance to skin fatty acids.     

Gastrointestinal carriage of methicillin-resistant Staphylococcus aureus.

Nasal and rectal cultures were taken from all patients with methicillin-resistant Staphylococcus aureus identified on routine cultures obtained because of clinical indications. Of 117 patients studied over a 3-year period, 70 (60%) had rectal colonization and 62 (53%) had nasal colonization. Rectal colonization, probably reflecting gastrointestinal carriage, may be a source of transmission of methicillin-resistant S. aureus in hospitalized patients and may be difficult to eradicate.     

Emergence of homogeneously methicillin-resistant Staphylococcus aureus.

Forty-seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), collected between 1986 and 1990 from 29 institutions, were analyzed for susceptibility to various antibiotics. Twenty-six strains were homogeneously methicillin resistant (i.e., greater than or equal to 10% of the cells in these strains were able to grow on Mueller-Hinton agar containing 50 micrograms of methicillin per ml). The MICs of gentamicin, clindamycin, trimethoprimsulfamethoxazole, methicillin, and imipenem for homogeneous MRSA strains were higher than those for heterogeneously resistant strains. Both types of strains were, for the most part, susceptible to vancomycin and trimethoprim-sulfamethoxazole. Ciprofloxacin-resistant MRSA strains were not isolated prior to 1988 but made up 40% of the post-1987 strains. The level of methicillin resistance correlated well with the imipenem MIC, suggesting that susceptibility to imipenem may serve as a marker to identify and monitor the prevalence of homogeneous MRSA strains.     

Molecular genotyping of methicillin-resistant Staphylococcus aureus strains in a teaching hospital in Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of nosocomial infections in our hospital. Therefore, we aimed to characterize MRSA isolates phenotypically from patients with nosocomial infections at Cumhuriyet University Hospital between December, 1999, and June, 2001, in Sivas by analysis of antibiotic patterns and genotypically using pulsed-field gel electrophoresis (PFGE) and repetitive element sequence-based polymerase chain reaction (rep-PCR). Forty-three nosocomial isolates were collected from various wards. All isolates were resistant to penicillin, tetracycline, oxacillin, and gentamicin. By rep-PCR and by separation of SmaI fragments of genomic DNA using PFGE, one major type (eight subtypes with PFGE) was identified among the strains. This clone was found to be different than some clones such as Iberian, Brazilian, and a major clone that was found in another Turkish University Hospital in Ankara. According to our results, there is a major MRSA clone with a potential to spread in our hospital. Infection control measures should be directed toward restricting the further spread of this clone. Therefore, in accordance with these findings, a surveillance culturing program should be established.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Community-acquired methicillin-resistant Staphylococcus aureus in Taiwan.

Staphylococcus aureus is a major cause of infections in both hospitals and communities, and is exhibiting increasing resistance to methicillin (methicillin-resistant S. aureus, MRSA) and related beta-lactams. MRSA is usually considered a nosocomial pathogen, but increasingly it is acquired in the community. In Taiwan, MRSA was colonized in a substantial proportion of healthy children and accounted for 25% to 75% of childhood community-acquired (CA) S. aureus infections. From the preliminary data, the isolates of sequence type (ST) 59 by multilocus sequence typing method appeared to be the major clone of CA-MRSA in northern Taiwan. Compared with those reported from the US and other countries, CA-MRSA isolates in Taiwan did not always harbor type IV staphylococcal cassette chromosome (SCCmec) and were resistant to multiple non-beta-lactam antibiotics, including clindamycin and macrolides. Molecular evidence suggested transmission of the community strain of MRSA into the hospital setting, and that the community strain had became a health care-associated pathogen. The treatment of putative CA S. aureus infection should be stratified according to the severity and the disease entity.     

Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays

This study evaluated a DNA oligonucleotide array that recognised 38 different Staphylococcus aureus targets, including all relevant resistance determinants and some toxins and species-specific controls. A new method for labelling sample DNA, based on a linear multiplex amplification that incorporated biotin-labelled dUTP into the amplicon, was established, and allowed detection of hybridisation of the amplicons to the array with an enzymic precipitation reaction. The whole assay was validated by hybridisations with a panel of reference strains and cloned specific PCR products of all targets. To evaluate performance under routine conditions, the assay was used to test 100 methicillin-resistant S. aureus (MRSA) isolates collected from a university hospital in Saxony, Germany. The results showed a high correlation with conventional susceptibility data. The ermA and ermC macrolide resistance genes were found in 40% and 32% of the isolates, respectively. The most prevalent aminoglycoside resistance gene was aphA3 (57% of the isolates), followed by aacA-aphD (29%) and aadD (29%); tet genes, mupR and dfrA were rare. There were no isolates with van genes or genes involved in resistance to quinupristin-dalfopristin. Enterotoxins were detected in 27% of the isolates. Genes encoding Panton-Valentine leukocidin, toxic shock syndrome toxin and exfoliative toxins were not found. The DNA array facilitated rapid and reliable detection of resistance determinants and toxins under conditions used in a routine laboratory and has the potential to be used for array-based high-throughput screening.     

Gastrointestinal colonization by methicillin-resistant Staphylococcus aureus in immunosuppressed mice.

ICR mice were inoculated intranasally with methicillin-resistant Staphylococcus aureus (MRSA) N133, and the inoculated MRSA was quantitatively recovered from the ceca and feces. The viable counts of the MRSA recovered from ceca correlated well with those from feces. Some mice eliminated MRSA from the cecum by 14 days after inoculation. Intraperitoneal administration of cyclophosphamide at a dose of 200 mg/kg 3 days before inoculation inhibited the elimination of the MRSA from both ceca and feces. All mice treated with cyclophosphamide excreted more than 10(4) CFU of the MRSA per g of feces for at least 70 days, indicating persistent colonization of the MRSA in the gastrointestinal tract. Some beta-lactam antibiotics decreased the colonization level, but others did not. The colonization level was suggested to depend on the antibacterial activity of the antibiotic against the MRSA and the degree of disturbance of intestinal flora by the antibiotic.     

Susceptibility of methicillin-resistant Staphylococcus aureus to lysostaphin.

One hundred and eleven isolates of methicillin-resistant Staphylococcus aureus recovered from patients at the Olin E. Teague Veterans Center from March 1983 to April 1987 were as susceptible to lysis by lysostaphin as methicillin-susceptible S. aureus controls were.     

Staff carriage of epidemic methicillin-resistant Staphylococcus aureus.

Twenty-six nurses were repeatedly screened for carriage of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) immediately before and after duty periods in which they solely attended six patients widely colonized with two EMRSA strains distinguishable by plasmid analysis. EMRSA carriage was detected in 13 nurses. Three EMRSA carriage patterns emerged: transient carriage in 12 nurses, when the EMRSA was isolated from noses or fingers of nurses after duty but was gone before their next day's duty; short-term nasal carriage, seen on occasion in 4 of these 12 nurses, when EMRSA carriage was detected on two consecutive screens; and persistent nasal carriage, seen in 1 nurse only, when the EMRSA was seen on more than two consecutive occasions. All but one of these incidents of carriage could be explained by close patient, rather than environmental, exposure and occurred despite an intensive control programme. Transient or short-term carriage in nurses probably resulted in transfer of the EMRSA between patients. Staff decontamination should be considered following a period of cohort nursing of EMRSA patients, especially if staff members are shortly to nurse unaffected patients. Our findings may explain some of the difficulties in controlling EMRSA.     

Fingerprinting methicillin-resistant Staphylococcus aureus by the immunoblot technique

A series of 133 isolates of methicillin-resistant Staphylococcus aureus was fingerprinted by the immunoblot technique. Extracts were prepared by lysostaphin degradation of overnight cultures and peptides were separated by SDS polyacrylamide gel electrophoresis. The peptides were transblotted on to nitrocellulose membranes and probed with (1) a hyperimmune rabbit serum raised against a methicillin-resistant S. aureus isolate, (2) a hyperimmune rabbit serum raised against an isolate of S. epidermidis, and (3) serum from a patient who had recovered from an infection with a methicillin-resistant S. aureus. This typing method confirmed the existence of an epidemic strain that accounted for 102 of the isolates. The remaining 31 isolates were grouped into a further seven types which correlated with the results of phage typing and antibiograms.     

Atopic dermatitis complicated by methicillin-resistant Staphylococcus aureus infection

INTRODUCTION: Staphylococcus aureus colonization/infection is commonly associated with disease severity in children with atopic dermatitis. The present report is a three-year retrospective chart review of five cases (comprising three boys and two girls, aged 9-15 years at referral) of methicillin-resistant S. aureus (MRSA) in children with moderate-to-severe atopic dermatitis. The review period spanned 2004-2007. All had longstanding severe disease, high IgE and eosinophil counts. Generalized erythema and a peculiar fishy odor were frequently observed by parents and physicians when MRSA was isolated during some of the episodes of exacerbation. All had tried various combinations of topical and systemic steroids, topical immunomodulants, traditional Chinese medicine and courses of antibiotics-without lasting relief. All specimens of MRSA had in-vitro sensitivity to vancomycin, with corresponding clinical correlates of disappearance of the erythema and fishy odor. CONCLUSION: A fishy odor and facial/generalized erythema in a patient with atopic dermatitis should alert the physician to screen for MRSA. The organism is rarely isolated, even among children with moderate-to-severe atopic dermatitis, and is usually sensitive to vancomycin.