Polymerase Chain Reaction-based DNA Fingerprinting Research Articles

Donor-Derived Metastatic Melanoma in a Liver Transplant Recipient Established by DNA Fingerprinting

Metastatic melanoma is a donor-derived malignancy that has rarely been reported in liver allograft recipients. We present a case of a transmitted donor-derived melanoma to a liver allograft recipient in whom the diagnosis was established by polymerase chain reaction-based DNA fingerprinting. A 52-year-old African-American man underwent a successful orthotropic liver transplant for alcohol-induced cirrhosis. One year after the orthotropic liver transplant, he presented at our institution with diffuse abdominal pain, and a computed tomography scan of the abdomen and chest showed innumerable masses diffusely involving the liver and multiple subcutaneous nodules in the abdominal and chest wall. A liver biopsy confirmed the diagnosis of metastatic melanoma. The origin of melanoma was traced to the donor by DNA fingerprinting of the native liver, the donor liver, and the donor gallbladder. Chemotherapy was initiated with temozolomide (75 mg/m² daily) and thalidomide (50 mg daily), to which he responded within 8 weeks with radiologic improvement in metastatic lesions. Tacrolimus was switched to sirolimus because of renal insufficiency as well as reported effectiveness against melanoma. Our patient survived for 9 months after the diagnosis of metastatic melanoma. He ultimately died of brain metastases. Donor-derived metastatic melanoma is a rare cancer with the highest transmission and mortality rates, which requires better recognition. Prompt diagnosis of donor-derived melanoma is critical and can be achieved reliably with polymerase chain reaction-based DNA analysis. Management options after diagnosis include de-escalation of immunosuppression, with or without urgent organ removal or retransplant. The roles of chemotherapy, immunotherapy, and radiotherapy require further study.

Molecular Typing of Streptococcus uberis Strains Isolated from Cases of Bovine Mastitis

The discriminatory power of two polymerase chain reaction-based DNA fingerprinting methods, random amplified polymorphic DNA and repetitive extragenic palindrome were compared by subtyping 128 isolates of Streptococcus uberis cultured from cows in six different dairy herds in New Zealand. The typing results demonstrated that the majority of isolates possessed unique fingerprint profiles except on occasions where multiple isolates were obtained from individual cows. On these occasions, individual quarters of the mammary gland were generally, but not exclusively, infected by the same strain of bacteria. Both random amplified polymorphic DNA and repetitive extragenic palindromic typing assays were simple to perform, relatively inexpensive ($11.00 per reaction), and provided reliable and reproducible results. Furthermore, when these assays were used in conjunction with each other, they provided a means of confirmation of the specific DNA fingerprint patterns obtained.

Outbreak investigation of nosocomial enterobacter cloacae bacteraemia in a neonatal intensive care unit.

Over a period of 7 months, 23 patients hospitalized in a neonatal intensive care unit (NICU) developed nosocomial Enterobacter cloacae bacteraemia. Contaminated saline for preparing heparin solution was initially identified as the common source of E. cloacae bacteraemia. Although environmental sanitation was enforced, the outbreak continued. E. cloacae has always been isolated from various cultures of the environmental specimens, from the hands of personnel and from the faeces of patients. All of the 23 bacteraemic isolates and 8 stool isolates from infected infants, as well as the 17 isolates from environmental specimens were found to be of the same genotype using the polymerase chain reaction-based DNA fingerprinting method. After various infection control methods were instituted, the outbreak eventually came under control. For epidemiological investigation, 23 neonates without E. cloacae bacteraemia were matched for case-control study. Nineteen (83%) of the case-patients were premature. The significant risk factors leading to E. cloacae bacteraemia in the NICU included small gestation age, low birthweight, exposure to personnel with contaminated hands and the presence of E. cloacae in the stool carriage (p=0.003, 0.007, 0.018 and 0.040, respectively). The gastrointestinal tracts of the patients and environmental surfaces appeared to be the principal sites of bacterial reservoir. In conclusion, the outbreak of E. cloacae bacteraemia was caused by a particular strain and possibly via multiple modes of transmission, including a bottle of contaminated saline as an initial common source, endogenous spread from the gastrointestinal tract and successive cross-infections between patients, hands of personnel and the environment. Effective infection control requires a multidisciplinary approach and reinforcement of infection control procedures, including aseptic technique, hand washing, proper isolation and disinfection of environmental surfaces.

Pyrolysis mass spectrometry analysis of free-living and symbiotic cyanobacteria.

The potential of pyrolysis mass spectrometry to distinguish closely related cyanobacterial strains was assessed by using the technique to compare symbiotic cyanobacteria isolated from the hornwort Phaeoceros laevis and free-living cyanobacterial strains at the same field site. The same strains had previously been compared using polymerase chain reaction-based DNA fingerprinting techniques (West & Adams 1997, Appl. Environ. Microbiol. 63: 4479-4484). Many of the strains were grouped identically by the two techniques, although there were some differences, possibly resulting from the ability of these cyanobacteria to develop a range of specialised cell types having different chemical compositions to the vegetative cells. Although growth conditions were chosen to suppress cellular differentiation, this may not always have been completely successful. With careful control of growth conditions pyrolysis mass spectrometry has considerable potential as an additional tool for the phenetic comparison of cyanobacterial strains. It has the advantage that analysis is directly derived from whole cells, and hence is simpler and cheaper than DNA-based methods, although it does require the growth of axenic strains. The technique may be particularly useful in the study of some of the more cryptic unicellular and non-heterocystous filamentous cyanobacterial groups, in which the lack of cellular differentiation should minimise any variability in the chemical composition of cells.

Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting

Polymerase chain reaction-based DNA fingerprinting was used as a tool to differentiate new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections (IMI) in dairy cows. The same subtype of S. uberis or S. dysgalactiae was detected from some infected mammary glands from one lactation to the next documenting the persistence of these infections. Conversely, some streptococci isolated from mammary glands during a lactation or from one lactation to the next were different subtypes suggesting that a new IMI occurred. These new streptococcal IMI would never have been detected using phenotypic methods of streptococcal identification. Results of this study indicate that PCR-based DNA fingerprinting can be used as an effective procedure to differentiate new and persistent S. uberis and S. dysgalactiae IMI in dairy cows. This technique will be useful in epidemiological investigations, and drug and vaccine efficacy studies when attempting to delineate new and persistent IMI.

Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting.

Polymerase chain reaction-based DNA fingerprinting was used as a tool to differentiate new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections (IMI) in dairy cows. The same subtype of S. uberis or S. dysgalactiae was detected from some infected mammary glands from one lactation to the next documenting the persistence of these infections. Conversely, some streptococci isolated from mammary glands during a lactation or from one lactation to the next were different subtypes suggesting that a new IMI occurred. These new streptococcal IMI would never have been detected using phenotypic methods of streptococcal identification. Results of this study indicate that PCR-based DNA fingerprinting can be used as an effective procedure to differentiate new and persistent S. uberis and S. dysgalactiae IMI in dairy cows. This technique will be useful in epidemiological investigations, and drug and vaccine efficacy studies when attempting to delineate new and persistent IMI.

Subtyping of Streptococcus dysgalactiae and Streptococcus uberis isolated from bovine mammary secretions by DNA fingerprinting.

Streptococcus dysgalactiae and Streptococcus uberis isolated from mammary secretions of cows from Tennessee and New Zealand were subtyped using polymerase chain reaction-based DNA fingerprinting. Such DNA fingerprinting using primer 8.6d (5'-GTAACGCC3') resulted in categorizing 116 S. dysgalactiae isolates into 25 different subtypes, with 17 subtypes observed in isolates from Tennessee and eight in isolates from New Zealand. All S. dysgalactiae DNA fingerprint profiles, regardless of origin, contained 700- and 330-base pair fragments. The majority of S. dysgalactiae isolates (73%) from Tennessee belonged to two subtypes. The remaining 23 isolates belonged to 15 different DNA fingerprint subtypes. Streptococcus dysgalactiae isolates from New Zealand (n = 32) were grouped into eight different subtypes; 66% belonged to two subtypes. A characteristic feature of S. dysgalactiae isolates from New Zealand was the presence of a 270-base pair DNA fragment seen infrequently in S. dysgalactiae isolates from Tennessee. When primer OPE-4 (5'-GTGACATGCC-3') was used, DNA fingerprinting differentiated S. uberis from Tennessee (n = 28) and New Zealand (n = 30) into 20 subtypes; 14 subtypes were observed in isolates from Tennessee, and six in isolates from New Zealand. All S. uberis DNA fingerprint profiles, regardless of origin, contained 1100-, 640-, and 450-base pair fragments. A characteristic feature of S. uberis isolates from New Zealand was the presence of a 300-base pair DNA fragment seen infrequently in S. uberis isolates from Tennessee. The most common subtypes of S. dysgalactiae and S. uberis from Tennessee herds were isolated in milk from lactating cows during monthly herd surveys, in milk from cows with clinical mastitis, and in mammary secretions from cows during the periparturient period, and thus were not confined to one particular stage of lactation. These data suggest that S. dysgalactiae and S. uberis from New Zealand are distinct from those isolated from the USA, and that DNA fingerprinting can be used as an epidemiological tool to differentiate streptococci and identify important sources of these mastitis pathogens on dairy farms.

Identification and Differentiation of Coagulase-Negative Staphylococcus aureus by Polymerase Chain Reaction

Staphylococcus aureus is an etiological agent of a wide variety of human and animal infections. The majority of S. aureus are coagulase-positive; however, some may be atypical in that they do not produce coagulase. Incorrect identification of an isolate can impact implementation of effective treatment and/or control measures. In this study, polymerase chain reaction-based DNA fingerprinting was used to differentiate coagulase-positive Staphylococcus aureus (CPSA) from coagulase-negative Staphylococcus aureus (CNSA). A total of 29 CNSA and 50 CPSA were evaluated. PCR-based DNA fingerprinting differentiated CNSA from CPSA on the basis of visible observation and densitometric evaluation. The method is rapid and accurate, eliminating variability associated with conventional techniques.

Application of Randomly Amplified Polymorphic DNA Fingerprinting for Species Identification of Bacteria Isolated from Bovine Milk

A polymerase chain reaction-based DNA fingerprinting system for species identification of bacteria in milk was developed using randomly amplified polymorphic DNA. A total of 108 organisms including 24 ATCC reference strains and 84 wild-type isolates belonging to gram-negative, Staphylococcus, Enterococcus, and Streptococcus species were used to develop the system. Organisms included in the study were those that are isolated frequently from milk. Forty primers from two commercially available primer kits were evaluated to determine the “ideal” primer that could be used for several bacterial species. Over 960 DNA fingerprint patterns were analyzed by laser densitometry. Seven of the 40 primers met criteria established for primer selection. However, only primers OPE-4 (5′ GTGACATGCC-3′) and OPE-20 (5′-AACGGTGACC-3′) allowed differentiation between all 19 ATCC bacterial species included in the study. The other five primers were restricted to either gram-negative bacteria (OPA-7, OPA-14), Staphylococcus species (OPA-13, OPA-14, OPA-18), or Streptococcus species (OPA-3). Primers OPE-4 and OPE-20 were further evaluated using 84 wild-type isolates. A bacterial species identification scheme was developed based on characteristic polymorphic DNA fragments obtained with primers OPE-4 and OPE-20. Results of this study suggest that RAPD fingerprinting has the potential for being developed into a rapid and accurate method for species identification of bacteria in milk.

Polymerase chain reaction-based DNA fingerprinting of Enterococcus faecium isolated from intramammary infections of dairy cows

Twenty of 71 cows in a dairy herd that calved between 1 November 1990 and 31 January 1991 had clinical or subclinical mastitis associated with Enterococcus faecium during early lactation. Forty-two E. faecium isolates were cultured from samples of mammary secretions collected from these 20 cows during this period. In the following calving season between 1 November 1991 and 31 January 1992, five isolates of E. faecium were identified from mammary secretion of four of 85 cows. All four cows had E. faecium intramammary infections within 14 days after calving. Isolates were subtyped using a polymerase chain reaction (PCR)-based DNA fingerprinting method. In addition, plasmid analysis was performed. PCR-based DNA fingerprinting differentiated the 47 isolates into four distinct subtypes. Five of six clinical mastitis isolates of E. faecium belonged to Subtype 2, and E. faecium Subtype 1 was the only subtype that was isolated from both calving seasons. PCR-based DNA fingerprinting was useful in tracing an infection of the same subtype in a cow over a period of time. New intramammary infections by different subtypes were identified also. Two plasmids of 4.8- and 5.1-kb were observed in seven strains identified as Subtype 3 by PCR-based DNA fingerprinting. Results of this study suggest that E. faecium can be an important cause of mastitis during early lactation. PCR-based DNA fingerprinting was useful for subtyping E. faecium isolates. Use of this technique could aid in understanding the epidemiology of bovine mastitis and facilitate development of more effective methods to control intramammary infections in dairy cows.

Polymerase Chain Reaction-Based DNA Fingerprinting for Identification of Streptococcus and Enterococcus Species Isolated from Bovine Milk

A total of 353 isolates including 304 isolates from bovine milk belonging to 8 Streptococcus and Enterococcus species, 14 American Type Culture Collection reference strains, 10 isolates from bovine milk that had atypical biochemical profiles, and 25 bovine milk isolates from Vermont were examined by polymerase chain reaction-based DNA fingerprinting. A single short oligonucleotide primer of eight bases (5′-GTAACGCC-3′) was used to produce a characteristic spectrum of amplified DNA fragments. Amplified DNA fragments were visualized following agarose gel electrophoresis and analyzed by scanning laser densitometry. Amplified DNA fragments were categorized as either primary, secondary, or variable fragments. DNA fragments observed in all isolates within a species were designated as primary [optical density > 0.3 absorbance units (AU)] and secondary fragments (optical density > 0.15 AU), while infrequent DNA fragments were designated as variable fragments (optical density >0.15 AU). Examination of fingerprint patterns revealed that each of the species evaluated possessed a unique fingerprint profile. A simple identification scheme was developed based on the occurrence of primary and secondary DNA fragments for a species. The polymerase chain reaction-based DNA fingerprinting assay was simple to perform, reliable, and reproducible. Results of this study indicate that DNA fingerprinting using polymerase chain reaction has the potential to be developed as a routine method for species identification of bacteria.

Genomic fingerprints of Staphylococcus aureus of bovine origin by polymerase chain reaction-based DNA fingerprinting.

Staphylococcus aureus (n = 75) isolated from mammary secretions of cows with subclinical and clinical mastitis from several geographic locations in the USA were examined using polymerase chain reaction-based DNA fingerprinting. DNA fingerprints were produced using a synthetic oligonucleotide primer (5'GTAACGCC3') to produce a distinct spectrum of amplified DNA fragments facilitating a high degree of resolution for differentiating S. aureus strains. PCR-based DNA fingerprinting grouped the 75 S. aureus isolates into 19 distinct profiles. The technique differentiated closely related strains within and between geographic locations. Findings suggest that certain types are found across geographic regions suggesting a common clonal type. Within herd data suggest heterogeneity among subclinical and clinical isolates of S. aureus strains. Compared to existing typing methods, PCR-based DNA fingerprinting is easy to perform and interpret. Use of PCR-based DNA fingerprinting may allow for a more detailed investigation of the epidemiology of S. aureus mastitis in dairy cows.

Differentiation of Mycobacterium tuberculosis strains by arbitrarily primed polymerase chain reaction-based DNA fingerprinting.

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of clinical isolates of M. tuberculosis strains. The primer which is specific to human papilloma virus (HPV) type 18 was found to be appropriate for the AP-PCR-based differentiation of M. tuberculosis isolates, since AP-PCR produced multiple polymorphic DNA bands when M. tuberculosis DNA was used as template. AP-PCR was performed using either one of the HPV type 18 primer and IS6110-specific primer (half-specific PCR, HS-PCR) or HPV type 18 primer pair only (nonspecific PCR, NS-PCR). The usefulness of these two methods in differentiating M. tuberculosis isolates, was measured by calculating dissimilarity values of 16 isolates using Cluster Analysis software. The highest dissimilarity values by HS-PCR and NS-PCR methods were 0.38 and 0.59, respectively. This suggested that NS-PCR method is better than HS-PCR method in strain differentiation. Although the dissimilarity value calculated by Cluster analysis of the standard restriction fragment length polymorphism method, in which IS6110 was used as a probe, was much more higher than the NS-PCR method, NS-PCR method using HPV 18 primers was quite useful for the differentiation of M. tuberculosis strains due to its rapidity and simplicity.