Pulsed-field Gel Electrophoretic Patterns Research Articles

Salmonella Enteritidis in Meat, Poultry, and Pasteurized Egg Products Regulated by the U.S. Food Safety and Inspection Service, 1998 through 2003

The U.S. Food Safety and Inspection Service (FSIS) tests for Salmonella in meat, poultry, and egg products through three regulatory testing programs: the Pathogen Reduction–Hazard Analysis and Critical Control Point (PR-HACCP) program, the ready-to-eat program for meat and poultry products, and the pasteurized egg products program. From 1998 through 2003, 293,938 samples collected for these testing programs were analyzed for the presence of Salmonella enterica serotypes. Of these samples, 12,699 (4.3%) were positive for Salmonella, and 167 (1.3%) of the positive samples (0.06% of all samples) contained Salmonella Enteritidis. The highest incidence of Salmonella Enteritidis was observed in ground chicken PR-HACCP samples (8 of 1,722 samples, 0.46%), and the lowest was found in steer-heifer PR-HACCP samples (0 of 12,835 samples). Salmonella Enteritidis isolates were characterized by phage type, pulsed-field gel electrophoretic pattern, and antimicrobial susceptibility. Phage typing of 94 Salmonella Enteritidis isolates identified PT13 (39 isolates) and PT8 (36 isolates) as the most common types. One isolate from a ready-to-eat ham product was characterized as PT4. Electrophoretic analysis of 148 Salmonella Enteritidis isolates indicated genetic diversity among the isolates, with 28 unique XbaI electrophoretic patterns identified. Of these 148 isolates, 136 (92%) were susceptible to each of 16 antimicrobials tested. Two isolates were resistant to ampicillin alone, and 10 isolates were resistant to two or more antimicrobials. Isolation of Salmonella Enteritidis from FSIS-regulated products emphasizes the need for continued consumer education on proper food handling and cooking practices and continued work to decrease the prevalence of Salmonella in meat, poultry, and pasteurized egg products.

Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish: a multicentre case-control study

Laribacter hongkongensis has been recovered from several patients with gastroenteritis. However, the causative role of this organism in human gastroenteritis is still unproven, and sources of the bacterium are unknown. We undertook a multicentre case-control study to investigate the association of L hongkongensis with gastroenteritis. Faecal samples from patients with community-acquired gastroenteritis and controls were cultured for L hongkongensis. Targeted food surveillance was done to identify potential sources of this bacterium. All isolates of this organism from patients and food items were characterised by pulsed-field gel electrophoresis and ribotyping. During a 4-month period, L hongkongensis was recovered from 17 of 3788 patients with community-acquired gastroenteritis, but was absent in 1894 controls (p=0.001). Those who were culture-positive for this bacterium had a recent history of travel (ten [59%] patients vs two [6%] of 34 matched controls, p<0.0001), of fish consumption (16 [94%] vs 19 [56%], p=0.009), and of eating minced freshwater fish meat (five [29%] vs one [3%], p=0.012). We recovered 25 L hongkongensis isolates from intestinal samples of freshwater fish and two from minced freshwater fish meat. Bacteria with the same pulsed-field gel electrophoretic pattern and ribotype were recovered from one patient and a sample of minced freshwater fish meat, which was from the same retail market recently visited by the patient. We did not see this particular combination of electrophoretic pattern and ribotype in any other isolates. L hongkongensis is associated with community-acquired gastroenteritis and traveller's diarrhoea. However, its causative role has not been shown. Freshwater fish is one source of this bacterium.

Phenotypic and molecular characterization of community occurring, Western Samoan phage pattern methicillin-resistant Staphylococcus aureus

In New Zealand, it is estimated that greater than half of the methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients belong to what has been termed Western Samoan phage pattern types 1 and 2 (WSPP1, WSPP2). These strains differ from classical MRSA isolates in terms of their lack of multiresistance and community occurrence, suggesting that such strains possess properties and/or characteristics different from those of other MRSA. To address this hypothesis, 10 WSPP1 and WSPP2 isolates from Western Samoa, New Zealand and Australia were compared with common hospital MRSA isolates. All WSPP isolates were identical with regard to pulsed-field gel electrophoretic pattern of SmaI-digested DNA, coagulase gene restriction fragment length polymorphism pattern and localization of mecA to a 194 kb SmaI digestion fragment. The WSPP strains were no more resistant/sensitive to various environmental stresses (e.g. skin fatty acids, UV light, desiccation) compared with hospital epidemic MRSA strains, except for their higher tolerance to salt. In terms of virulence, the WSPP MRSA were quantitatively better at attaching to the epithelial cell line HEp2, were uniformly egg-yolk opacity factor negative and produced higher levels of haemolytic toxins compared with non-WSPP MRSA isolates.

An outbreak of diarrhoea due to multiple antimicrobial-resistant Shiga toxin-producing Escherichia coli O26:H11 in a nursery.

An outbreak due to Shiga toxin-producing Escherichia coli O26:H11 (STEC) occurred at a nursery in southeastern Japan in 1997. Thirty-two children had watery or bloody diarrhoea but none of them suffered from haemolytic-uremic syndrome. All of the STEC O26 were isolated during the period from 23 July to 22 August from 24 children, 3 nurses, and 2 food samples. These organisms had stx1 and eae genes but none of the other genes for which we tested (stx2, bfp, and EAF plasmid). They also possessed multiple antimicrobial resistances, which were encoded by a transmissible plasmid, and showed mostly identical genomic pulsed-field gel electrophoretic patterns. The results of this investigation suggested that contaminated food was the main contributing factor to this multiple antimicrobial-resistant STEC O26 infection, and person-to-person transmission also contributed to the spread of this outbreak.

Septic Arthritis Following Arthroscopic Meniscus Repair: A Cluster of Three Cases

Summary: Three cases of Staphylococcus epidermidis septic arthritis following inside-out arthroscopic meniscus repair within a 4-day period at the same facility are described. All three patients responded to surgical debridement and 4 to 6 weeks of intravenous antibiotics. In each instance, the meniscus and repair sutures were left intact; 12- to 38-month follow-up revealed no evidence of infection or meniscal symptoms. Epidemiological investigation implicated the meniscus repair cannulas as one of the few factors common to all three cases. Molecular typing of bacterial DNA revealed that two of the three isolated organisms showed identical pulsed-field gel electrophoretic patterns, implying a common source of inoculation. Experimental contamination of the cannulas revealed that only sterilization involving ultrasonification, lumen washing by water jet, and steam sterilization resulted in clean and sterile cannulas.0749-8063/99/1501-1800$3.00/0

Type E botulism associated with vacuum-packaged hot-smoked whitefish

On January 16, 1997 two Germans got botulism after eating hot-smoked Canadian whitefish produced in Finland. The serum sample of one of the patients contained 6 MLD/ml of botulinum toxin. The type of toxin was identified as E by the toxin neutralization test and the botulinum neurotoxin type E (BoNT/E) gene was also amplified from the serum by polymerase chain reaction (PCR), but C. botulinum could not be isolated from the positive serum sample. The remains of the hot-smoked whitefish eaten by the patients contained botulinum toxin detected by the mouse bioassay and the BoNT/E gene as determined by PCR. C. botulinum was isolated from the fish sample and it was confirmed to be type E by the mouse bioassay and by PCR. Eleven other fish samples from the same lot did not contain botulinum toxin nor any BoNT gene. The incriminated food was processed on the 9th and 10th of January, 1997 from frozen whitefish imported to Finland from Canada. The pulsed-field gel electrophoretic pattern of the isolated C. botulinum strain resembled a reference strain of North American origin. It did not match any C. botulinum strains isolated from the Baltic sea-bottom or from the fish caught in the area indicating that the fish was contaminated by C. botulinum in Canada. The conditions resulting in toxin production could not be identified. The safety problems associated with vacuum-packaged hot-smoked fish seem to be of utmost concern and the product is one of the most important botulism food vehicles processed on an industrial scale. Temperature monitoring and the use of time–temperature indicators are to be recommended in order to ensure adequate storage temperature from processing through to consumption. Allowing the use of nitrate and nitrite together with sufficiently high NaCl concentration in this particular product should also be considered.

Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo.

Several multidrug-resistant (MDR) isolates of Streptococcus pneumoniae recovered from a variety of clinical sources and expressing serotypes 9N, 14, 19F, and 3 could not be distinguished from the capsular type 23 Spanish/USA epidemic clone in antibiotype, pulsed-field gel electrophoretic pattern, and restriction fragment length polymorphism types of their penicillin binding protein genes 1A, 2X, and 2B. When tested in a mouse model of virulence, isolates expressing capsular type 3 were lethal for 100% of infected animals within 2 days after intraperitoneal injection of 10(2) cfu/mouse. In contrast, several capsular type 23F isolates belonging to the MDR Spanish/USA clone and recovered from the same site were not lethal, even when injected at the dose of 10(7) cfu/mouse. These data suggest that the pneumococcal isolates expressing serotypes 9N, 14, 19F, and 3 represent products of in vivo capsular transformation events in which the MDR epidemic capsular type 23F Spanish/USA clone was the recipient.

Phenotypic resistance to thrombin-induced platelet microbicidal protein in vitro is correlated with enhanced virulence in experimental endocarditis due to Staphylococcus aureus.

Thrombin-induced platelet microbicidal protein (tPMP) is secreted by rabbit platelets following thrombin stimulation, and it kills common endovascular pathogens in vitro, including Staphylococcus aureus. Therefore, pathogens which exhibit tPMP resistance in vitro possess a potential survival advantage in vivo at sites of endovascular damage. We generated an isogenic S. aureus strain pair, differing in tPMP susceptibility, by transposon (Tn551) mutagenesis of a tPMP-susceptible (tPMPs) parental strain (ISP479) to derive a stably tPMP-resistant (tPMPr) strain, ISP479R. ISP479 and ISP479R were equivalent in vitro in the following phenotypes: biotyping, antiobiograms, platelet adherence and aggregation, growth kinetics, cell wall-associated protein A expression, and fibrinogen binding. Genotypic comparisons of chromosomal DNA of strains ISP479 and ISP479R following restriction endonuclease digestion revealed indistinguishable pulsed-field gel electrophoretic patterns. The genotype exhibited by strain ISP479R was linked to the tPMP-resistant phenotype, as it was transducible into the initially tPMP-susceptible parental strain, ISP479. Southern hybridization verified the presence of a single copy of Tn551 in the same chromosomal restriction site of both ISP479R and tPMPr transductants of ISP479. The correlation of in vitro tPMP susceptibility phenotypes with the ability to induce experimental endocarditis (a prototypical endovascular infection) was evaluated. Despite equivalent rates of endocarditis induction, animals infected with strain ISP479R achieved significantly higher vegetation bacterial densities over a 7-day post-challenge period than did animals infected with strain ISP479. These data suggest that tPMPr microbial strains have a selective advantage in experimental staphylococcal endocarditis. Furthermore, the major impact of tPMP resistance upon endocarditis pathogenesis appears to involve a postvalvular adherence event(s), most probably by facilitating bacterial proliferation within vegetations.

Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea.

Thirty-eight antibiotic-resistant isolates of Streptococcus pneumoniae recovered in a hospital in Seoul, Korea, between February 1990 and March 1992 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of SmaI chromosomal digests. Most of the isolates were from sputum samples, and a few strains were from otitis media and meningitis. The great majority of isolates (34 of 38, or 89%) were multiresistant, sharing virtually identical, elevated minimal inhibitory concentration (MIC) values (microgram/ml) for penicillin (1-2), chloramphenicol (12-25), tetracycline (25-30), and sulfamethoxazole/trimethoprim (> 100). Twenty of the isolates were also resistant to erythromycin, and all isolates were also considered to be resistant to ceftriaxone and cefotaxime (1-2) according to the new breakpoint definitions. The most frequent serotypes were 23F (17 of 38) and 19F(14 of 38); 2 belonged to serotype 15B and 1 of 24F. Of the remaining 4 isolates (2 serotype 3, 1 type 6B, and 1 type 9V) all were resistant to tetracycline and sulfamethoxazole/trimethoprim and with the exception of 1 of the type 3 strains, were also resistant to chloramphenicol. Eleven of the 14 serotype 19F isolates shared a relatively homogeneous PFGE pattern, which was indistinguishable from the PFGE pattern shown by most (12 of 17) of the 23F isolates. The PFGE pattern of these 19F and 23F isolates was also indistinguishable from the PFGE pattern shown by representative multiresistant capsular type 23F isolates from Croatia, Portugal, and New York City and the findings document the extensive geographic spread of this multidrug-resistant S. pneumoniae clone. The data also suggests in vivo capsular transformation of the multiresistant clone from serotype 23F to serotype 19F.

Methicillin-resistant Staphylococcus aureus isolates recovered from a New York City hospital: analysis by molecular fingerprinting techniques.

Fifty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates collected at New York Hospital Medical Center of Queens in 1989 were analyzed by molecular fingerprinting techniques. Close to 70% of these isolates (38 of 55) shared a common pulsed-field gel electrophoretic pattern, carried the same mecA gene polymorph type II, were free of the transposon Tn554, and would not react with a mecI-specific gene probe. An additional five isolates shared all properties of the major MRSA clone except that they carried mecA gene polymorph type III. All these isolates had an extremely heterogeneous methicillin resistance phenotype that belonged to population analysis profile class 1 or 2. The rest of the 12 MRSA isolates showed a variety of chromosomal pulsed-field gel electrophoretic patterns that carried different mecA polymorphs and that also gave positive reactions with DNA probes for Tn554 and for the mecI gene. The molecular features of the majority MRSA clone suggest that it is an archaic MRSA isolate similar in features to early MRSA isolates recovered in the 1960s.

Two new leptospiral serovars in the Hebdomadis serogroup isolated from Zimbabwe cattle.

Four strains belonging to the genus Leptospira serogroup Hebdomadis were isolated from Zimbabwe cattle at slaughter. These isolates were subjected to cross-agglutinin absorption tests and to restriction fragment length polymorphism and pulsed-field gel electrophoresis analyses of their genomic DNAs. One of these strains represents a new serovar, for which the name mhou is proposed; strain SBF 40 is the reference strain of this serovar. The other three strains belong to a second new serovar, for which the name marondera is proposed; the reference strain of this serovar is strain SBF 5. The three strains of serovar marondera could be differentiated by their restriction fragment polymorphism and pulsed-field gel electrophoretic patterns.

Virulence and genotype stability ofSalmonella entericaserovar Berta during a natural outbreak

Strains of Salmonella enterica serotype Berta, collected over a period of 6 years from a well documented natural outbreak in Denmark, have been characterized in order to assess the stability of chromosomal typing systems and virulence properties. Outbreak strains were identical in Pvu II and PSTI IS200 profiles, all but two strains showed the same Sma I ribotype, and all but one strain showed the same Not I pulsed field gel electrophoretic pattern, indicating that these molecular markers remained almost constant during the outbreak. In general, strains of S. Berta were found to be of moderate to low virulence; log VC10 values were found to vary between 3.0 and 4.4 after i.p. challenge of mice, and maximum CFU in internal organs of day-old chicks varied between 2 and 4 log10 units following oral challenge. The minor differences observed between strains in vivo did not correlate with differences in in vitro invasion into cultured MDCK cells, nor with in vitro growth characteristics. A succession of different plasmid profile types was observed during the outbreak but a hierarchical selection of clones based on differences in virulence was unlikely to have caused the succession of types of S. Berta during this outbreak.

Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid-1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin-susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefânia and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade.

Resolution of clonal subgroups among Neisseria gonorrhoeae IB-2 and IB-6 serovars by pulsed-field gel electrophoresis.

Analysis of macrorestriction patterns by PFGE to resolve the relatedness of clonal subgroups amongst N gonorrhoeae IB-2 and IB-6 serovar strains. Nineteen IB-2 and eight IB-6 serovar strains that differed in either auxotype or penicillin sensitivity were isolated over a two and a half-year period from patients attending several STD clinics in Sydney. During this period, a major clone, Wt/IB-2 (FS), established on epidemiological grounds, was circulating amongst homosexual males. The genetic relation of this major clone to the other strains present in the community was determined by pulsed-field gel electrophoretic (PFGE) analysis of DNA restriction fragments. Genomic DNA from the 27 isolates were prepared, digested with SpeI and BglII and the restriction patterns were analysed by contour-clamped homogeneous electric field electrophoresis (CHEF) in a CHEF DRIII equipment. Phenotypic characterisation of the 27 isolates by the combined use of auxotype, serological characterisation and penicillin sensitivity indicated the presence of subgroups within each of the two serovars. In the present study, PFGE analysis of SPeI and BglII-generated genomic DNA restriction patterns from six of the ten Wt/IB-2 (FS) correlated well with phenotypic characterisation of this major clone. Four of the ten Wt/IB-2 (FS) were found to be clonally-derived variants of this major clone as minor genome variations (less than 3 DNA fragments) were observed. Distinct clones were represented by three Wt/IB-2 (LS) isolates as the DNA fingerprints generated from these were unrelated to the major clone. Analysis of PFGE patterns of 6 Pro/IB-2 isolates showed that one was genotypically identical to the major clone, two were clonal variants and three had significantly different patterns to indicate that they were genotypically unrelated. Wt/IB-6 isolates had heterogenous PFGE patterns that were clearly unrelated to the Wt/IB-2 serovar strains. Within the IB-6 serovar, there were three isolates with the Wt/IB-6 (FS) phenotype that could be considered as clonal variants whilst the rest were genotypically distinct. PFGE analysis of macrorestriction patterns generated from SpeI- and BglII-cleavage of genomic DNA has enabled the establishment of clonal origins of strains present in the Sydney community during the period of study. The delineation of strains belonging to major A/S groups by PFGE analysis presents a clearer epidemiological picture than phenotypic characterisation alone.

Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia.

Seventy-two penicillin-resistant Streptococcus pneumoniae isolates collected from clinical specimens in the Czech and Slovakian Republics between 1990 and 1992 were analyzed by a variety of molecular techniques. Most of the highly resistant isolates (40/72) (penicillin MIC between 1 up to 16 micrograms/ml) were represented by two distinct pneumococcal clones, and most of these isolates (35/40) were also resistant to at least two other antibiotics (tetracycline plus chloramphenicol or erythromycin). All 17 isolates belonging to the first clone were of serotype 14, had very high penicillin MICs (8-12 micrograms/ml), shared a common, abnormal penicillin-binding protein (PBP) pattern and one of two related pulsed-field gel electrophoretic (PFGE) patterns. The 15 isolates belonging to the second clone were all of serotype 19A, had penicillin MICs between 1 and 4 micrograms/ml, shared a unique, abnormal PBP pattern, and could be divided into two subgroups on the basis of PFGE patterns, one of which was indistinguishable from the PFGE pattern of a multiresistant capsular type 19A clone of S. pneumoniae already identified earlier in Hungary. Thirty-two of the 72 pneumococcal isolates had lower penicillin MICs (0.1-0.5 microgram/ml), and these isolates differed from the more highly resistant ones in several respects: They belonged to seven different serotypes, showed large variation in PFGE patterns (20 patterns in 32 isolates) and most of them (21/32) were resistant to penicillin only. Tentative explanations for these findings, in terms of epidemiological and molecular mechanisms, are considered.

Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a pediatric hospital in Zagreb, Croatia.

Sixty-four penicillin-resistant Streptococcus pneumoniae isolates [benzylpenicillin minimal inhibitory concentrations (MICs) between 0.05 and 1.6 micrograms/ml] recovered at the Pediatric Hospital "Dr. Fran Mihaljevic" in Zagreb, Croatia between October 1990 and March 1993 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of chromosomal DNA fragmented by digestion with the SmaI endonuclease. Hospital "Dr. Fran Mihaljevic" services the capital of Croatia and its vicinity. Most of the isolates were from nasopharyngeal carriage, but several isolates were from otitis media, sinusitis, and meningitis. Most isolates belonged to either serotype 23F (36/64) or 19F (12/64); the rest, including three 15C isolates, were in 11 additional distinct serotypes. The overwhelming majority (25/36) of the serotype 23F isolates had penicillin MIC values of 1-2 micrograms/ml and shared variants of a common PFGE pattern, closely related to the PFGE identified in multiresistant pneumococci of the same serotype with wide geographic spread to Spain, Portugal, France, and the United States. This group of bacteria was also resistant to tetracycline, chloramphenicol, and sulfamethoxazole/trimethoprim. In contrast to the relative genetic and phenotypic homogeneity of the more highly penicillin resistant isolates, pneumococci with penicillin MICs between 0.5 and 0.4 microgram/ml (29/64) were distributed in 13 different serotypes and as many as 20 distinct PFGE patterns.

Spread and maintenance of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone during an outbreak of MRSA disease in a Spanish hospital.

It was not until November 1989 that the 1,000-bed University-affiliated Hospital de Bellvitge "Princeps d'Espanya" in Barcelona first acquired methicillin-resistant Staphylococcus aureus (MRSA). Since that time, the outbreak of MRSA disease has continued. We have analyzed by genomic DNA fingerprinting 189 MRSA isolates collected between late 1989 and the end of 1993. The isolates include both invasive and colonizing strains as well as isolates from health-care workers and environmental sources. In addition, 52 clinical isolates of methicillin-susceptible S. aureus (MSSA) collected in the same hospital were also analyzed. Isolates were classified into clonal types on the basis of molecular typing techniques. A single MRSA clone (I::B::a) belonging to ClaI type I, pulsed-field gel electrophoretic pattern B, and Tn554 pattern a was responsible for the great majority of infections (73% of blood cultures and 79% of specimens from other clinical sources). This clone appeared at the very beginning of the outbreak, spread throughout the hospital wards, and was also carried by inpatients and health-care workers and on environmental surfaces. In contrast, no dominant lineage was apparent among MSSA isolates (33 distinct pulsed-field gel electrophoretic patterns among 52 isolates). Two MSSA isolates seem to have originated from the dominant clone by deletion of the mecA gene and some additional DNA. In several isolates, different mecA polymorphs were present in identical chromosomal backgrounds or cells with distinct chromosomal backgrounds carried the same mecA polymorph, suggesting horizontal transfer of the mecA gene.