Identical Pulsed-field Gel Electrophoresis Profiles Research Articles

Variable number tandem repeat loci providing discrimination within widespread genotypes of Acinetobacter baumannii

Some genotypes of Acinetobacter baumannii, defined by pulsed-field gel electrophoresis (PFGE), have been found in many hospitals. Our aim was to find variable number tandem repeat (VNTR) loci capable of providing discrimination among isolates with highly similar or identical PFGE profiles, to gain insights into the epidemiology. Thirteen loci identified in A. baumannii ATCC 17978 were tested using a panel of isolates that included multiple representatives of genotypes belonging to the three European clonal lineages. Two loci, with repeat units of 9 and 6 bp respectively were selected. Repeat numbers varied between 3 and 29, and 9 and 26 respectively at the two loci. The repeat numbers of representatives of each genotype often differed between hospitals, providing a means of tracking patient transfers and possible transmissions between patients. The results suggest that this analysis accurately reflects the known epidemiological information, and provides a valuable tool for cross-infection studies.

Hospital outbreak of Burkholderia stabilis bacteraemia related to contaminated chlorhexidine in haematological malignancy patients with indwelling catheters

Burkholderia cepacia complex (BCC) is an opportunistic pathogen that occasionally causes hospital outbreaks. This paper describes an outbreak of BCC bacteraemia in haematological malignancy patients related to a contaminated chlorhexidine gluconate solution. Eight BCC isolates were obtained from patients hospitalised in the same ward of a cancer centre in a Korean hospital. A further three BCC isolates were obtained from 0.5% chlorhexidine gluconate used in the same ward. The isolates were identified as B. stabilis and exhibited identical pulsed-field gel electrophoresis profiles. All patients with B. stabilis bacteraemia had indwelling intravenous catheters, which were treated with chlorhexidine to disinfect the catheters. Following identification of the source of contamination, strict controls regarding surveillance cultures for disinfectants have been enforced. No further B. stabilis infections have been found in the hospital.

Persistence of Salmonella Senftenberg in poultry production environments and investigation of its resistance to desiccation

Most Salmonella serovars, including Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg), are tolerant to desiccation and able to colonize and persist in feed mills. In addition, they may survive cleaning and disinfection procedures used on poultry farms. The present study was conducted to investigate the survival of S. Senftenberg in broiler parent stock farms and broiler farms. The isolates from one of the parent stock farms investigated only differed by a single band in fluorescent amplified fragment-length polymorphism analysis and had identical pulsed-field gel electrophoresis profiles, indicating that a S. Senftenberg clone had persisted for more than 2 years, despite cleaning, disinfection, desiccation and depopulation, and was subsequently able to infect Salmonella-free layers. Isolates from the same house on a different broiler parent stock farm were found to be identical by amplified fragment-length polymorphism analysis and pulsed-field gel electrophoresis although the farm tested negative for Salmonella 55 times over a period of 18 months between the two positive samplings. An assay was developed to investigate the survival of 34 S. Senftenberg isolates during desiccation at approximately 38% relative humidity. On average, the viability of S. Senftenberg isolates decreased by 1000-fold over 35 days. The persistent clones were no more resistant to desiccation than the other isolates investigated. However, S. Senftenberg was more resistant to desiccation than an isolate of Pantoea agglomerans commonly found on poultry feed-processing lines. This study demonstrates the risk of persistence of feed-associated serovars such as S. Senftenberg.

Genetic Diversity among Clonal Lineages withinEscherichia coliO157:H7 Stepwise Evolutionary Model

Escherichia coli O157:H7 variants were examined for trait mutations and by molecular subtyping to better define clonal complexes postulated on the O157:H7 evolution model. Strains of beta-glucuronidase-positive, sorbitol-negative O157:H7 isolated in United States and Japan were identical to A5 clonal strain and shared sequence type (ST)-65 by multilocus sequence typing (MLST); thus, they belong in A5. However, these strains exhibited pulsed-field gel electrophoresis (PFGE) profile differences that suggested genomic divergence between populations. Sorbitol-fermenting O157 (SFO157) strains from Finland, Scotland, and Germany were identical to A4 clonal strain and belong in A4. Some SFO157 strains, isolated years apart and from different countries, had identical PFGE profiles, suggesting a common origin. Despite similarities, some Finnish and Scottish and all of the German strains have ST-75 ("German clone"), whereas others have ST-76, a new variant ("Scottish clone"). MLST of strains in other clonal complexes also discriminated strains thought to be identical and showed that genetic differences will further distinguish clonal populations into subclones.

Molecular epidemiology of meticillin-resistant coagulase-negative staphylococci in a Swedish county hospital: evidence of intra- and interhospital clonal spread

During a 10-month period in 2003, consecutive routine clinical cultures from patients treated in 15 different ward units in a Swedish county hospital were screened for the presence of meticillin-resistant coagulase-negative staphylococci (CNS). Genetic similarity between isolates was analysed using pulsed-field gel electrophoresis (PFGE). The results were compared with multi-drug-resistant Staphylococcus epidermidis isolated previously at the tertiary referral hospital. In total, 428 isolates of CNS were identified, of which 188 (44%) were meticillin resistant. Three clusters (Groups A, B and C) of S. epidermidis, each consisting of more than 10 isolates, with a PFGE-DNA similarity of >90% were identified. The strains in Groups A and B (N=15 and N=13, respectively), which were generally resistant to gentamicin, co-trimoxazole and clindamycin, originated from 24 patients, of whom 21 had been treated in the intensive care unit (ICU) before sampling. The third cluster, Group C, involved 14 isolates from 14 patients. Only two of these patients had stayed at the ICU, and all for less than one day. Isolates in Group C were less resistant than those in Groups A and B. Isolates belonging to Group A showed an identical PFGE profile compared with multi-drug-resistant S. epidermidis isolated from patients at the referral hospital. This study demonstrated the persistence and spread of meticillin-resistant clones of CNS within the county hospital, especially in the ICU, and possible interhospital spread of a multi-drug-resistant clone between the county and referral hospitals.

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.

Clinical and environmental distribution of Legionella pneumophila in a university hospital in Italy: efficacy of ultraviolet disinfection

The molecular epidemiology of Legionella pneumophila in the 'V. Monaldi' University Hospital was studied. Seven cases of nosocomial Legionnaires' disease were diagnosed between 1999 and 2003. Two clinical legionella strains obtained from two patients in the adult cardiac surgery unit (CSU) and 30 environmental legionella strains from the paediatric and adult CSUs, neonatal intensive care unit (NICU) and the cardiorespiratory intensive care unit (CR-ICU) were serotyped and genotyped. L. pneumophila serogroup 1/Philadelphia with an identical pulsed-field gel electrophoresis (PFGE) profile A was isolated from two patients in the adult CSU, and from three and one water samples taken in the adult CSU and the paediatric CSU, respectively, from 2001 to 2002. Furthermore, L. pneumophila serogroup 3 with an identical PFGE profile B was identified in 20 environmental strains from all wards, L. pneumophila serogroup 3 with PFGE profile C was identified in a single environmental strain from the CR-ICU, and non-pneumophila Legionella with identical PFGE profile D was identified in five environmental strains from the adult CSU, paediatric CSU and NICU. Ultraviolet irradiation was effective in disinfection of the hospital water supplies in the adult and paediatric CSUs contaminated by L. pneumophila clone associated with nosocomial Legionnaires' disease. In conclusion, these data demonstrate that two cases of nosocomial legionellosis were caused by the persistence of a single clone of L. pneumophila serogroup 1/Philadelphia in the hospital environment, and that disinfection by ultraviolet irradiation may represent an effective measure to prevent nosocomial Legionnaires' disease.

Molecular epidemiology of Salmonella spp. isolates from gulls, fish-meal factories, feed factories, animals and humans in Norway based on pulsed-field gel electrophoresis

The molecular epidemiology of 98 isolates of Salmonella serovar Agona (n = 27), S. Montevideo (n = 42) and S. Senftenberg (n = 29) from wild-living gulls, fish-meal factories, feed factories, humans and domestic animals was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis. Two of the S. Agona profiles were identified both in gulls and in two of the factories. In addition, one of these profiles was detected in two infected poultry farms. Two of the S. Montevideo profiles were also identified both in gulls and in two of the factories, and one of these profiles was observed in a human isolate. Four factories shared an identical S. Senftenberg profile. The S. Senftenberg profile found in gulls was not identified in any other source investigated. The presence of isolates with identical PFGE profiles indicates potential epidemiological links between different factories, as well as between gulls and factories.

Diversity and Host Range of Shiga Toxin-Encoding Phage

Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage. Toxin-encoding phages from C600::933W and from six clinical E. coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E. coli. The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed. Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups. The interaction between Shiga toxin-encoding phage and intestinal E. coli was examined. Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage. While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E. coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages. Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny. Different phages were found to lysogenize different strains of intestinal E. coli. Lysogeny was found to occur more commonly than lytic infection. The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E. coli O157:H7.

Outbreak of multi-resistant Klebsiella oxytoca involving strains with extended-spectrum beta-lactamases and strains with extended-spectrum activity of the chromosomal beta-lactamase.

This study was conducted to analyse broad-spectrum cephalosporin-resistant Klebsiella oxytoca strains. The 57 isolates studied were recovered from clinical specimens (n=23) or from rectal swabs (n=34) during a 26-month period. Antibiotic susceptibility patterns were determined using standard agar diffusion and dilution methods including the synergy test between extended-spectrum cephalosporins and clavulanic acid. ERIC-2 PCR and pulsed-field gel electrophoresis (PFGE) methods were used to study the clonal relatedness of the strains. Plasmid-mediated and chromosomal beta-lactamases were characterized by mating and specific bla gene amplification and sequencing. Four different antibiotic resistance patterns were identified whereas ERIC-2 PCR and PFGE revealed six main profiles. Extended-spectrum beta-lactamases (ESBLs) were found in 32 strains: TEM-7 (n=26), TEM-129 (n=1), TEM-3 (n=4), SHV-2 (n=1). The new TEM-type beta-lactamase, TEM-129, differed from TEM-7 by one mutation (Glu-104-->Lys). All TEM-7 or TEM-129 producers were genetically related. Twenty-five other strains with identical ERIC-2 PCR and PFGE profiles harboured a bla(OXY-2) gene different from the reference gene: 24 strains displayed one substitution (Ala-237-->Ser) in the KTG motif and one strain, highly resistant to ceftazidime, showed an additional substitution (Pro-167-->Ser). The study demonstrated that the majority of strains (n=52) harbouring the OXY-2-type beta-lactamase corresponded to two clones. The first clone (n=27) corresponded to ESBL-producing strains. The second clone (n=25) displayed extended-spectrum activity of the chromosomal beta-lactamase.

Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks

Three food poisoning restaurant outbreaks due to Staphylococcus aureus, occurring during June–October 2002 in the Principality of Asturias (PA), Spain, provided the basis for investigating some aspects of the molecular epidemiology of this organism. The methods applied to identify strains and lineages included multiplex-polymerase chain reaction (PCR) to detect nine enterotoxin ( se) genes, and three DNA fingerprinting procedures: pulsed-field gel electrophoresis (PFGE) with SmaI, randomly amplified polymorphic DNA (RAPD) with two selected primers, and plasmid restriction analysis with HindIII. Thirty-two isolates were differentiated into three non- se and 12 se strains, which were outbreak-specific, except for one that was represented in two of the outbreaks. In outbreak 1, the 16 food isolates analyzed had sec, seg and sei genes and generated a distinctive DNA fingerprint, being assigned to a single strain. This strain could be categorized as endemic in the PA and associated to manually handled dairy products and nasal carriers. In outbreak 2, the four food isolates analyzed fell into three strains, each one displaying a different se-gene profile ( sea, sec and seg-seh-sei) and a distinctive DNA fingerprint. In outbreak 3, the five food isolates tested fell into four seg-sei strains generating identical RAPD but different PFGE and plasmid profiles, and one sea strain also collected from two nasal carriers. This last strain had also been found in manually handled vegetables in outbreak 2, and it belongs to a not very frequently found sea lineage in the PA. Multiplex-PCR to detect se genes together with the three applied DNA fingerprint typing procedures proved therefore to be useful tools in subclassifying S. aureus for epidemiological purposes.

Community outbreak of perianal group A streptococcal infection in Denmark.

Perianal group A streptococcal infection (PASI) occurs primarily in children. There is limited information on the incidence, transmission and treatment of PASI. We report a cluster of cases connected to a Danish kindergarten and observations of the incidence of PASI in the local population. A Danish rural community with 1765 children 15 years and younger registered with two general practice clinics. After being alerted of a possible cluster of PASI cases, all isolates of group A beta-hemolytic streptococci were collected and subjected to T typing and pulsed field gel electrophoresis (PFGE) if grown from either a rectal swab or an accompanying throat swab obtained in the offices of local general practitioners during the ensuing 4-month period. Clinical data were obtained from the files of the local general practitioners. Twelve cases of PASI were caused by group A beta-hemolytic streptococci T type 28 with an identical PFGE profile: 6 of the cases were in children attending the same kindergarten, 4 were connected otherwise to the cluster and 2 cases seemed to be unrelated. Five cases of PASI with different T types and PFGE profiles were diagnosed during the same period giving an estimated annual incidence of 2 to 7 per 1000 children. Penicillin V was ineffective in 3 cases, and no recurrence was seen after change of the treatment to oral clarithromycin. A clone of T type 28 seemed to be the cause of the largest cluster of PASI cases described thus far. Clarithromycin was effective as second line treatment. An estimated annual baseline incidence of 2 to 7 per 1000 in the local population indicates that PASI may not be as rare as previously estimated.

Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources in Calcutta, India

Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing Escherichia coli (STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were stx(1) and stx(2) (44.5% of strains) and stx(1), stx(2), and hlyA (enterohemorrhagic E. coli hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored eae. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India.

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae ( M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type ( M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38 T) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles. Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1 T) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.

Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-field gel electrophoresis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.

Epidemiological typing of bovine streptococci by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to investigate the epidemiology of streptococcal mastitis in dairy cattle. The most prevalent streptococcal species, Streptococcus uberis (60-80% of streptococcal isolates), was highly heterogeneous, with different cows only rarely sharing the same pulsotype. S. agalactiae was rarely encountered, however all eight isolates from one farm generated identical PFGE profiles, which differed from those of all other isolates examined, confirming cow-to-cow transmission. Fifty-two isolates of S. dysgalactiae from 27 cows on 5 farms generated 6 different profiles. However, on individual farms, only one or two pulsotypes usually predominated. This species is generally regarded as an environmental pathogen but our data suggest that cow-to-cow transmission of S. dysgalactiae may occur. In spite of the variation in PFGE profiles of isolates from different cows, persistent infections in individual cows were usually caused by the same pulsotype of S. uberis or S. dysgalactiae.

Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India.

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.