Predominant Pulsed-field Gel Electrophoresis Types Research Articles

Molecular tracking of Listeria monocytogenes in an Iberian pig abattoir and processing plant

The environment and products from an Iberian pig abattoir and processing plant were sampled to investigate the prevalence and genetic diversity of Listeria monocytogenes. The organism was not detected in the pig carcasses prior to processing. Fresh and dry-cured pork did show detectable levels, always ranging below 100 CFU per gram. A total of 163 L. monocytogenes isolates collected during one year were characterized by PCR-based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. Three predominant PFGE types or pulsotypes seemed to persist in the plant. The pulsotype S1 (serotype group 1/2a, 53% of the isolates) was mostly recovered from whole pieces of meat and environmental sites, while pulsotypes S2 (1/2a, 17%) and S4 (1/2b, 21%) were more frequently associated with ground pork products. The pulsotype S4 was also found in a grinding machine, suggesting a possible association of this machine with the contamination of the ground meat products.

Virulence genes of bovineStaphylococcus aureusfrom persistent and nonpersistent intramammary infections with different clinical characteristics

To screen putative virulence genes in Staphylococcus aureus causing persistent and nonpersistent bovine intramammary infections (IMI) with different clinical characteristics. To examine, whether a possible relationship exists between genetic profile and infection persistence, clinical signs of infection, clonal type determined by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. One-hundred and sixty-one S. aureus isolates derived from bovine IMI, consisting of 17 different PFGE types, were screened by conventional and multiplex-polymerase chain reaction (PCR) for 24 virulence genes for haemolysins (hla-hlg), leukocidins (lukED, lukM), exfoliative toxins (eta, etb), enterotoxins (sea-seo, seu), toxic-shock syndrome toxin (tst), and genes encoding penicillin (blaZ) and methicillin resistance (mecA). The majority of S. aureus isolated at the onset of mastitis carried haemolysin genes (76.7-97.4%), lukED (96.6%), and at least one gene for pyrogenic toxin superantigen (PTSAg) (69.0%). Strains carrying PTSAg-encoding genes were more common among predominant PFGE types and in persistent IMI. Strains concomitantly possessing sed, sej, and blaZ, putatively plasmid-encoded, were typically found in connection with persistent IMI. Our results suggest that certain genetic elements are over-representative in S. aureus isolates especially from persistent bovine mastitis. This phenomenon seems to be in connection with clonal type and is often concomitant with penicillin resistance. This is the first study to investigate associations between a large number of bacterial factors and outcome of S. aureus mastitis. The finding that widespread clonal types of S. aureus causing bovine mastitis of low treatment response may harbour characteristic genes could be improved for strain-specific diagnostic purposes.

Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from Broiler Poultry and Pig Farms in England and Wales

This study aimed to investigate the occurrence and molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) isolates on poultry and pig farms in England and Wales. A total of 217 VREF isolates were obtained from fresh feces and environmental swabs collected from conventional and organic farms. A predominant pulsed-field gel electrophoresis (PFGE) profile was found for each VREF-positive farm, together with less frequent types. All isolates presented the vanA genotype and were esp negative. Seventy-six percent of the VREF isolates were additionally resistant to nine or more antimicrobials, presenting a diverse range of resistance phenotypes. The multiresistance traits did not appear to be specific to individual farms or sample types (i.e., environmental or fecal), nor did they correlate with any specific PFGE type. Ninety-three percent of the isolates were resistant to penicillin, 89% were resistant to tetracycline, 87.5% were resistant to erythromycin, and 50% were resistant to quinupristin-dalfospristin (Synercid). The lack of clonality among these populations may suggest the horizontal transfer of resistance genes and/or a dynamic replacement of clonal lines rather than persistence.

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.

Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk.

The duration of infection and pulsed-field gel electrophoresis (PFGE) types of bovine intramammary Streptococcus uberis isolates were examined. Milk samples were collected in duplicate from all 4 glands of 503 cows from 5 herds within 1 to 3 d of parturition and from 113 cows with clinical mastitis in the same herds throughout lactation. Glands from which S. uberis was isolated were resampled at 28-d intervals.The prevalence of S. uberis was 12% for cows around parturition, and the median duration of infection was 16 d. Cows >2 yr old had a longer duration of infection than 2 yr old cows, and duration varied among herds. A total of 173 different PFGE types were identified from a total of 234 S. uberis isolates. Each farm had a unique set of PFGE types. Only 3 PFGE types were common to each of 3 pairs of cows, and these occurred on the same farm. Where S. uberis was isolated on more than one occasion from a gland, only 55% of the PFGE types were the same across time. For cows with multiple glands infected, only one-half (9 of 18) had the same PFGE type in more than one gland. No predominant PFGE type was identified in any herd. It is concluded that there was wide heterogeneity of PFGE types, that the environment rather than other cows was the likely source of S. uberis infections, and that glands may be infected with multiple S. uberis PFGE types over a lactation.

Characterization of isolates of methicillin-resistant Staphylococcus aureus from Hong Kong by phage typing, pulsed-field gel electrophoresis, and fluorescent amplified-fragment length polymorphism analysis.

The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major "clones" at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.

A major methicillin-resistant Staphylococcus aureus clone predominates in Malaysian hospitals.

This study was conducted to determine the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysian hospitals. A total of 264 MRSA isolates from eight hospitals were subjected to typing by pulsed-field gel electrophoresis (PFGE) of SmaI restricted DNA. Antibiotic disk susceptibility testing was also carried out to determine their resistance patterns. Thirty-one PFGE pattern types were identified. Three major pattern types A, ZC and K were found with type A the predominant profile in c. 80% of strains and present in all hospitals. Unlike type A, other DNA pattern types were unique to the hospitals in which they were isolated. PFGE type A also consisted of strains that were multiply antibiotic resistant. The presence of a single predominant PFGE type in Malaysian hospitals is an important finding which suggests that inter-hospital spread of MRSA had occurred frequently and regularly.

Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection.

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of S. sonnei isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S. sonnei predominantly (n = 59) from three counties in the west of Ireland. Phage typing (n = 17), plasmid profiling (n = 28), and integron analysis (n = 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A (n = 53) and PFGE type B (n = 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (n = 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among S. sonnei isolates circulating in this region. The limited diversity of the S. sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.

Detection of a novel repeated sequence useful for epidemiological typing of pathogenic Yersinia enterocolitica

AbstractStrains (n=203) of Yersinia species were used in genotyping and PCR experiments in order to evaluate the genotyping potential of the YeO:3RS probe. This probe comprises a 12.5 kb genomic fragment of the Y. enterocolitica O:3 lipopolysaccharide O-antigen gene cluster cloned into plasmid pBR322. The genotyping potential of YeO:3RS was shown to reside in the region upstream of the O-antigen gene cluster, i.e., in the first 1.65 kb of the cloned genomic fragment that contains a repeated sequence (RS) present in multiple copies in the genome. In genotyping, the YeO:3RS probe was hybridised to DNA of Yersinia enterocolitica isolates (n=112) from humans, animals and food, along with strains of other Yersinia species (n=5) and Salmonella enterica strains (n=3). The YeO:3RS probe efficiently detected and subtyped all European pathogenic Yersinia enterocolitica isolates of the serobiotypes O:3/4, O:9/2 and O:5,27/2 studied (n=87), whereas it hybridised only weakly or not at all with the other strains. Within Yersinia enterocolitica serobiotype O:3/4 strains, YeO:3RS genotyping was as discriminatory as genotyping by pulsed-field gel electrophoresis (PFGE) of XbaI-NotI digested genomic DNA. When these two methods were combined, YeO:3RS genotyping divided both of the two predominant PFGE types into six subtypes, thus increasing the discrimination. In PCR screening of additional 86 Yersinia strains, the 1.65 kb region was detected in European pathogenic serotypes O:1 and O:2 in addition to serotypes O:3, O:5,27 and O:9, indicating that it can be exploited in detecting and typing of European pathogenic serotypes in general.

The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in the United States, 1994-2000.

The genetic relatedness of 672 penicillin-resistant isolates of Streptococcus pneumoniae (PRSP) recovered during national surveillance studies conducted in the United States during the periods of 1994-1995, 1997-1998, and 1999-2000 was determined by use of pulsed-field gel electrophoresis (PFGE). Overall, 104 different PFGE types were elucidated. For all study periods combined, the 12 most prevalent PFGE types included >75% of all isolates, and 5 types were closely related to widespread clones (Spain(23F)-1, France(9V)-3, Spain(6B)-2, Tennessee(23F)-4, and Taiwan(19F)-14). From 1994-1995 to 1999-2000, 3 major PFGE types (not closely related to 16 recognized clones) increased in prevalence. Multidrug resistance was identified among 96%-100% of the isolates in 9 of 12 predominant PFGE types. The prevalence of erythromycin resistance increased within 4 major PFGE types. These observations support the hypothesis that the dominant factor in the emergence of PRSP in the United States during the 1990s has been human-to-human spread of relatively few clonal groups that harbor resistance determinants to multiple classes of antibiotics.

Typing of human Campylobacter jejuni isolates in Finland by pulsed-field gel electrophoresis.

A total of 69 pulsed-field gel electrophoresis (PFGE) types were identified among 176 Campylobacter jejuni isolates from Finnish patients. In two geographic areas studied, five predominant PFGE types comprised over 40% of the isolates. One-third of the isolates had unique PFGE types. In small outbreaks, identical PFGE patterns were demonstrated, indicating a common source of infection.