Plasmid Patterns Research Articles

Oxacillin- and Quinolone-Resistant Staphylococcus Aureus in Sao Paulo, Brazil: A Multicenter Molecular Epidemiology Study

To investigate the possibility of interhospital spread of multiresistant Staphylococcus aureus in Sao Paulo, Brazil. We evaluated 13 nosocomial S aureus strains selected because of resistance to oxacillin and ciprofloxacin. The strains were collected between March 1991 and September 1991 from four different hospitals in Sao Paulo. Two were teaching hospitals, and two were private hospitals. Each strain was isolated from a different patient. All patients were hospitalized when the strains were isolated. The strains were typed by restriction endonuclease analyses of plasmid DNA (REAP) using EcoRI, HindIII, RsaI, and AluI and by extended antibiogram profile (34 drugs). All strains had identical plasmid and antibiogram profile. They demonstrated the same plasmid pattern as previously described in one of the hospitals studied. Our results suggest the dissemination of a unique oxacillin- and quinolone-resistant strain of S aureus in several hospitals of Sao Paulo, Brazil.

Conformity and diversity among field isolates of Rhizobium leguminosarum bv. viciae, bv. trifolii, and bv. phaseoli revealed by DNA hybridization using chromosome and plasmid probes

A study was made of 113 bacterial isolates from root nodules of peas, lentils, red clover, and French beans, which had been grown in the same soil. Plasmid band profiles visualized in Eckhardt gels were analysed in relation to DNA hybridization patterns obtained by probing restricted total cellular DNA in Southern blots. Rhizobium leguminosarum chromosomal probes (plac12, pCOS309.1) and various symbiotic plasmid (nod gene region) probes were used. Dominant plasmid DNA hybridization patterns and more frequent combinations of plasmid patterns and chromosomal types were found among the isolates of each host plant species; the occurrence of alternative combinations indicated that genetic transfer and recombination among members of this soil population had taken place. About 40% of all isolates belonged to the same chromosomal type. Isolates of the same chromosomal type were often found with cryptic plasmids of the same size in different host plant species. Although isolates could not be assigned to their respective plant host groups using chromosomal probes alone, this was generally possible using symbiotic plasmid probes and the results were in complete accordance with plant tests. However, there was a group of bean isolates in which no homology to any of the R. leguminosarum probes was detected under the conditions of high stringency used. Other exceptional isolates of beans conformed in probe tests and subsequent plant host specificity tests better to biovars viciae or trifolii than to biovar phaseoli; thus, the nodulation of beans (i.e., Phaseolus vulgaris) in the field appears less subject to stringent control of specificity than that of other host plant species. It was also noted that the nod gene regions probed showed greater diversity in isolates of biovars viciae and trifolii than in biovar phaseoli.Key words: Rhizobium leguminosarum, genetic diversity, plasmid, DNA hybridization, restriction fragment length polymorphism.

Spread of Klebsiella pneumoniae producing SHV-5 beta-lactamase among hospitalized patients.

The first outbreak of infections caused by an SHV-5 producing strain of Klebsiella pneumoniae is reported. Within a period of 1 year and 9 months, multiresistant K. pneumoniae strains caused severe infections, mostly of the lower respiratory tract, in 22 patients. The strains were resistant to penicillins, third-generation cephalosporins, aztreonam, chloramphenicol, tetracycline and co-trimoxazole. The resistance determinants were transferable to Escherichia coli. All isolates produced a beta-lactamase with a pI of 8.2. Ceftazidime was hydrolyzed at this band. These characteristics, together with the resistance phenotype, are identical to those of a reference strain producing the beta-lactamase SHV-5. The K. pneumoniae strains of all patients were identical in their capsular serotype (K1), plasmid pattern and plasmid fingerprint after digestion with Dra I restriction endonuclease. We conclude that this outbreak was caused by the spread of one clone of K. pneumoniae producing SHV-5 beta-lactamase among patients of different wards. Our results indicate a real risk for failure of therapy by third-generation cephalosporins in intensive care patients due to SHV-5 producing pathogens.

Increased frequency of large R-plasmids in Klebsiella pneumoniae colonizing patients with spinal cord injury

From 1978 to 1988 strains of gentamicin-susceptible (Gm s ) and gentamicin-resistant (Gm r ) Klebsiella pneumoniae were saved from annual surveillance cultures of the perineal region of patients with spinal cord injury (SCI). Of 38 strains selected for further study (24 Gm s and 14 Gm r), there were 23 different serotypes (two nontypable). Fourteen Gm s as well as 14 Gm r strains displayed no common plasmid patterns, but all contained a large plasmid of 168–208 kb. Among the 14 Gm r strains, nine had large conjugative plasmids of approximately the same size (166–193 kb), which conferred to a susceptible Escherichia coli host an identical resistance pattern: ampicillin, chloramphenicol, gentamicin, piperacillin, trimethoprim-sulfamethoxazole, tetracycline, and tobramycin. Of the nine transconjugants, eight contained a single plasmid. One transconjugant contained a 168-and 80-kb plasmid. Restriction endonuclease digestion patterns of the R-plasmids revealed minimal similarity. We conclude that, during a 10-year period, different large R-plasmids have spread among multiple serotypes of K. pneumoniae in spinal cord injury (SCI) patients in one rehabilitation hospital. We hypothesize that other genes located on large, R-, and non-R-plasmids may confer an additional advantage for colonization by K. pneumoniae in SCI patients.

Isolation of a DNA probe for identification of glucan‐producing Pediococcus damnosus in wines

Some Pediococcus damnosus strains isolated from spoiled wines were able to synthesize polysaccharide and increased viscosity. Three strains lost their ropy phenotype relatively frequently during successive transfers in a non‐alcoholic medium. They could not recover it when cultured in the presence of ethanol. Plasmid pattern comparison showed these variants had lost some of their plasmid DNA. Both ethanol and the plasmid were required to induce polysaccharide synthesis.Plasmid DNA of a wild strain was cloned into Escherichia coli with pTZ18R and pBR 322. Specific clones were selected by differential hybridization of transformed E. coli with plasmid DNA from a ropy strain and its non‐ropy variant. Clones which hybridized with total plasmid DNA from the ropy strain and not with that of the non‐ropy strain carried an insert of the plasmid involved. One of these was chosen. Its DNA proved to be specific and usable as a probe in identifying and detecting ropy Ped. damnosus strains.

Plasmid Pattern Analysis of Staphylococcus Species Isolated from Bovine Mammary Secretions

Plasmid profiles of staphylococci isolated from bovine mammary secretions were heterogeneous as shown by the study of 94 isolates representing six species. Plasmids were identified in 19 of 94 staphylococcal isolates. Number of plasmids per isolate varied from 1 to 4. Size of plasmids ranged from 1.2 to 45 MDa; however, most were between 1.8 and 4.8 MDa. Some isolates with identical plasmid profiles were observed within and between species. Plasmid profiles observed in this study suggest that no specific plasmid pattern occurs within a species. Ability to differentiate isolates was not enhanced when antibiograms were used in conjunction with plasmid profiles. Plasmid pattern analysis does not appear to be an adequate method for discriminating between isolates of a species and would likely provide limited epidemiological information regarding staphylococci of bovine origin.

Plasmid-pattern analysis in the differentiation of Staphylococcus epidermidis isolates

Plasmids carried by 29 Staphylococcus epidermidis isolates from two different origins (invasive and carrier, non-hospital-associated strains) were analysed by agarose gel electrophoresis in order to evaluate any association with virulence. Plasmid patterns were found to be characteristic of individual strains and no particular molecular size band was found to correlate with either of the two categories of isolates. It is concluded that the plasmid pattern analysis carried out could not differentiate between carrier and invasive S. epidermidis, but did provide a useful tool for epidemiological investigation.

Identification by DNA sequence analysis of a new plasmid-encoded trimethoprim resistance gene in fecal Escherichia coli isolates from children in day-care centers.

In our ongoing studies of trimethoprim resistance (Tmpr) in day-care centers (DCC), we have shown a high rate of fecal colonization with Tmpr Escherichia coli and, using total plasmid content analysis, have shown that this is due to a diversity of strains. In the present study, we analyzed 367 highly Tmpr (MIC, greater than or equal to 2,000 micrograms/ml) isolates of E. coli from 72 children over a 5-month period and found at least 83 distinct plasmid patterns, indicating that at least 83 strains were involved. Several strains were particularly common in a given DCC, including one found in 61% of children with Tmpr E. coli; these common strains usually persisted within a DCC for several months. Colony lysates were hybridized with gene probes for dihydrofolate reductases (DHFR) types I, II, III, V, and VII; 21% hybridized under stringent conditions, and all of these were with type I (17%) or type V (4%) probes. Tmpr was cloned from a probe-negative Tmpr transconjugant, and an intragenic probe was prepared from this clone. Approximately 21% of the Tmpr E. coli strains (76 isolates) in the DCC were found to have this new gene, 74 of which were in one DCC. The DNA sequence of this gene was determined, and the predicted amino acid sequence was shown to have between 32% and 39% identity with the amino acid sequences for types I, III, V, VI, and VII and the partial sequence of type IV and approximately 26% identity with types IX and X DHFR. This confirms the uniqueness of this gene, which has tentatively been named dhfrxii, and its translation product, DHFR type XII.

Characterization of Azospirillum associated with maize (Zea mays) in France, using biochemical tests and plasmid profiles

Thirty Azospirillum strains were isolated from the rhizosphere of 13 maize (Zea mays L.) cultivars grown in 14 French soils, using a new specific method, which has been given the name ROSEA. Among these strains 26 were Azospirillum lipoferum and 4 were Azospirillum brasilense. Their characterization was achieved using biochemical tests and plasmid profiles. Biochemical patterns allowed clear differentiation between the two species. A large diversity in carbon source metabolism was found among the Azospirillum sp. strains regardless of their origin. The A. brasilense were much more closely related, and were found in only two of the rhizospheres studied. The 30 plasmid patterns were all different, and the plasmid-profiling technique can therefore be considered as strain specific. All the A. lipoferum harboured a 150-MDa plasmid, while all the A. brasilense harboured a 90 to 100-MDa plasmid. This result reinforces the hypothesis of the presence of such plasmids as an additional criterion for differentiating these two species. Key words: Zea mays, Azospirillum, ROSEA method, biochemical tests, plasmid profiles.

Differentiation of Listeria monocytogenes isolates by using plasmid profiling and multilocus enzyme electrophoresis

Three hundred and seven Listeria monocytogenes isolates from various origins (clinical sources, raw chicken, seafoods, dairy and meat products and processing environments) were screened for plasmids. The overall frequency of L. monocytogenes isolates containing plasmids was 77%. The highest percentages of plasmid positive isolates were found from meat (89%), chicken (81%) and dairy products (64%), while clinical isolates had the lowest plasmid percentage (28%). Seven sizes of plasmids (21, 24, 27, 35, 40, 47 and 52 MDa) were distinguished. All sizes were represented in the meat isolates, clinical isolates contained only two of the plasmid sizes, while several different sizes of plasmids were found in the isolates from other origins. Plasmid profiling divided the isolates into ten plasmid pattern types. Multilocus enzyme electrophoresis of 75 isolates demonstrated 12 distinctive multilocus genotypes (ETs) which clustered into two groups: cluster A including serotype 1 and 4 isolates, and isolates not typable by Difco antisera serotype 1 and 4, and cluster B containing only serotype 1 isolates. No relationship between ETs and plasmid profiles could be demonstrated.

Molecular characterization of strains of enteroinvasive Escherichia coli O143, including isolates from a large outbreak in Houston, Texas.

A large diarrhea outbreak due to enteroinvasive Escherichia coli (EIEC) serogroup O143 occurring in Houston, Tex., provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was done by comparing the plasmid patterns and the chromosomal restriction endonuclease digestion patterns by pulsed-field gel electrophoresis (PFGE) of EIEC from the outbreak, other E. coli from the same serogoup (O143), and EIEC isolated from other patients with diarrhea. Among the isolates studied, there was marked restriction fragment length polymorphism. All 3 non-O143 EIEC isolates had very different restriction endonuclease digestion patterns, as did 5 of 5 O143 non-EIEC isolates and 6 of 15 O143 EIEC isolates. Four Houston outbreak O143 EIEC isolates had the same restriction pattern as an O143 EIEC strain isolated 2 months before in Mexico and was nearly identical to another two O143 EIEC Mexican isolates. These related strains also had the same plasmid pattern; however, the presence of only a few plasmid bands, versus the 21 to 30 chromosomal bands seen with PFGE, suggests that plasmid patterns could be a less specific way to distinguish different strains. These results demonstrate that PFGE can distinguish between different E. coli strains of the same serogroup and phenotype. This technique can also identify relatedness within O143 EIEC, and our data suggest the spread of a strain of EIEC from Mexico to Houston, where it caused a large outbreak. PFGE may be useful to study the epidemiology of EIEC.

Effect of fermented oatmeal soup on the cholesterol level and the Lactobacillus colonization of rat intestinal mucosa.

Rats were fed with freeze-dried oatmeal soup fermented by six different Lactobacillus strains from rat and man; the formula is intended for enteral feeding. The serum cholesterol levels after 10 d were lower for rats eating oatmeal as compared to a commercial product, Biosorb Sond. Colonizing ability of the administered strains were evaluated in vivo. Only Lactobacillus reuteri R21c were able to, effectively, colonizing the mucosa; it represented about 30% of the Lactobacillus population 24 d after termination of the administration. L. reuteri R21c was easily recognized by the ability to produce a yellow pigment on agar plates. The identity was confirmed by carbohydrate fermentations (API 50CH), plasmid pattern and endonuclease restriction analysis of the chromosomal DNA.

Molecular epidemiology of coagulase-negative staphylococci isolated from immunocompromised patients.

To define the source of invasive coagulase-negative staphylococci (CNS) and the epidemiology of strain variation in immunocompromised patients. Weekly microbial surveillance cultures were obtained from the nares, throat, skin, rectum, and urine. Plasmid pattern analysis was performed on all coagulase-negative staphylococci isolated from blood cultures and on selected strains from the surveillance sites. A 902-bed, university-owned, tertiary-care referral hospital. Forty-four patients on the bone marrow transplant or hematologic malignancy services. Plasmid pattern analysis was performed on 340 surveillance isolates (median = 7 per patient) and 201 bloodstream isolates (median = 3 per patient). Patients were colonized with numerous unique strains (median = 5 per patient) of coagulase-negative staphylococci. The 44 patients had 108 episodes of positive blood cultures, 20 of which were preceded by colonization with the same strain. Isolation of the matching strain from surveillance cultures preceded the positive blood culture by 1 to 8 days in 9 episodes and 18 to 389 days in 11 episodes. The matching strain was isolated from the skin in only 6 (30%) of those episodes and from mucosal sites in 70%. Of the 108 episodes of positive blood cultures, 21 were identified as nosocomial bloodstream infections. Four of the 21 nosocomial bloodstream infections were preceded by colonization with the same strain. In all 4 episodes, the infecting strain was cultured from the nares before the blood cultures were obtained. Our results suggest that mucous membranes might be sources for strains of CNS causing bacteremia.

Intrahospital spread of a single gentamicin-resistant, beta-lactamase-producing strain of Enterococcus faecalis in Argentina.

Six beta-lactamase-producing (Bla+) isolates of Enterococcus faecalis recovered over a 17-month period from an Argentinian pediatric hospital were found to have identical or almost identical chromosomal restriction patterns by pulsed-field gel electrophoresis, although the plasmid patterns were different. These isolates, like Bla+ enterococci in the United States, hybridized to a staphylococcal Bla gene probe. The presence of a single strain was somewhat surprising, since all isolates transferred Bla by conjugation.

Nosocomial Staphylococcus epidermidis septicaemia among very low birth weight neonates in an intensive care unit

A cluster of cases of septicaemia caused by coagulase-negative staphylococci was observed among the infants at the Neonatal Intensive Care Unit (NICU) of the Karolinska Hospital in May 1987. The presence of a unique antibiogram among the blood culture isolates prompted an investigation to determine whether an epidemic strain existed or not, using antibiogram, biotyping and plasmid profiles as epidemiological markers. All 14 isolates with a unique antibiogram were investigated, and 22 isolates without the unique antibiogram served as controls. Of the 14 isolates, 11 were Staphylococcus epidermidis and had similar plasmid patterns. Of the 22 control isolates, 15 were S. epidermidis, and none had the special plasmid pattern nor any other recurring plasmid patterns. The use of plasmid profile analysis together with antibiograms thus identified a possible epidemic strain of S. epidermidis which may have been responsible for the upsurge of septicaemia cases at the NICU. The presence of an epidemic strain implies that hospital cross infection control could be important in preventing neonatal septicaemia caused by S. epidermidis.

Analysis of plasmid pattern in paediatric intensive care unit outbreaks of nosocomial infection due to Enterobacter cloacae

Sequential outbreaks of nosocomial infection due to multiply-resistant Enterobacter cloacae occurred in September 1987, and between December 1988 and January 1989, in a paediatric intensive care unit. A total of eight neonates were affected and most had received ventilatory support. Initially, we were unable to determine whether the two outbreaks were caused by the same strain of E. cloacae. After applying plasmid profile analysis to identify epidemic strains, we established that the strain from the first outbreak was different from the second outbreak strain, as each had its own plasmid pattern. During the second outbreak, an environmental bacteriological survey was carried out. We found that the distilled water containers were contaminated with E. cloacae which had the same plasmid profile. After changing the distilled water containers and by reinforcement of aseptic techniques, the nosocomial outbreak was terminated.

Plasmids in Bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance

Obligately thermophilic strains of Bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis. All strains in our collection contained large plasmids (20 × 10 6–80 × 10 6) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins. The major plasmid species were designated pSE407 (38.7 × 10 6), pSE409(29.0 × 10 6), pSE411 (21.5 × 10 6), and pSE410 (23.5 × 10 6). Their physical endonuclease maps were constructed, and by Southern blots and hybridizations it was shown that these plasmids were related. From curing experiments and electrotransformations (electroporations) we conclude that pSE407, pSE410, and pSE411 code for temperature resistance. In addition pSE410 codes for bacteriocin production and resistance. Plasmid pSE409 probably also codes for bacteriocin production and resistance.

Plasmids and phase variation in Xenorhabdus spp

Three strains of Xenorhabdus nematophilus (A24, F1, NC116) and strain Dan of Xenorhabdus bovienii were tested to evaluate whether the phase variation observed in these bacteria was in any way connected with plasmids. The plasmid patterns of both phases of A24 and F1 strains were the same, whereas the two NC116 phases had only one band each. No difference was observed between the undigested or digested plasmid patterns of the two phases from the three strains. No plasmid was detected in either phase of strain Dan. The plasmid probes were prepared from the six bands of A24 phase 1. By hybridization studies, three plasmids in two forms (open circular and supercoiled) were detected in the strain A24. Two were estimated at 12 kb, and the smallest was about 4 kb. Attempts to hybridize plasmid probes with either undigested or digested chromosomal DNA of the two phases of strain A24 were unsuccessful. The results suggest that neither a difference in plasmid content nor a plasmid recombination with the chromosome is involved in phase variation. The hybridizations revealed homologous DNA sequences among the three plasmids of strain A24 and among the plasmids of strains such as A24 and NC116, which were isolated from geographically distant countries, suggesting that plasmids may encode similar proteins.

Characterization by plasmid profile analysis of acetic acid bacteria from wine, spirit and cider acetators for industrial vinegar production

The fermentation in industrial high acid submerged fermenters of wine, spirit or cider into vinegar was investigated by direct analysis of the plasmid patterns of the acetic acid bacteria involved. The strains were different in all samples. One strain from a red wine acetator was isolated by the double layer agar technique and proved to be the main fermenting organism. After inoculation of an acetator with this strain, the initiation of acidification was accelerated. Inoculation with spirit vinegar organisms led to immediate acidification. The spirit vinegar organisms were outnumbered after 12 d by fortuitously‐occurring, highly adapted red wine vinegar bacteria. Over‐oxidation was due to physiological regulation, a genetical change by plasmid loss or occurrence of new organisms was not observed.

Molecular distribution of ROB-1 beta-lactamase in Actinobacillus pleuropneumoniae

The plasmid content and molecular distribution of the ROB-1 beta-lactamase was investigated in 31 swine isolates of Actinobacillus pleuropneumoniae. Three types of plasmid patterns were observed in ampicillin-resistant isolates: a predominant one consisting of 2.6-, 2.9-, and 5.0-kb plasmids and two other patterns consisting of 4.2- and 5.5-kb plasmids and of a 6.8-kb plasmid. Plasmid DNA preparations were hybridized with a blaROB-1 intragenic fragment from the Haemophilus influenzae Rrob plasmid. Positive hybridizations were observed with all ampicillin-resistant isolates. The blaROB-1 gene was found on 3 plasmids of 2.6, 5.5, and 6.8 kb. One swine isolate of Pasteurella multocida also had a 2.6-kb plasmid bearing blaROB-1. ROB-1 was confirmed in typical isolates by isoelectric focusing and blaROB-1 sequences were confirmed by polymerase chain reaction amplification with an intragenic set of primers. Plasmids bearing blaROB-1 were successfully electro-transformed in a susceptible A. pleuropneumoniae isolate. These results emphasize the importance of ROB-1 in A. pleuropneumoniae and identify a possible reservoir of beta-lactam resistance.