Genome analysis of five soil bacterial isolates named formerly Enterobacter agglomerans

The genomes of five nitrogen-fixing strains isolated from the vicinity of Bayreuth and named formerly Enterobacter agglomerans were studied and compared with the genomes of several Rahnella aquatilis strains as well as with one Pantoea agglomerans and one Ent. agglomerans reference strains, obtained from different world collections; they all were previously assumed to be related to this group of natural isolates.
By using the infrequently cutting restriction endonuclease XbaI , highly chracteristic fingerprints were obtained for each of the studied strains except two Ent. agglomerans isolates which had identical fingerprints. By hybridization of the resulting individual PFGE-fingerprints with a rDNA probe, containing the rrnB ribosomal RNA operon of Escherichia coli , the relationship between the analysed strains was studied. It was shown that the natural isolates are very closely related to the type strain of R. aquatilis —ATCC 33071. The genome sizes of all studied strains were estimated to be between 4.4 and 5.8 Mb on the basis of the lengths of their Xba I fragments. By a modification of the PFGE technique it was shown that the analysed strains harbour one to three large and extra large plasmids with sizes in the range 90 to 608 kb.     

Plasmids and genetic determinants of antibiotic resistance of gram-negative bacteria

Distribution of plasmids and genetic determinants of antibiotic resistance was studied in 129 strains of Pseudomonas, Klebsiella, Serratia and Enterobacter isolated from oncological patients. It was shown that 56 isolates contained the plasmids, 9 conjugative plasmids being plasmids with broad bacterial host spectrum. A significant part of the strains contained genes controlling production of APH (3"), type II APH (3'), type I and II DHPS and type type II DHFR. Genetic determinants of tetracycline resistance of classes D and E were detected for the first time in the strains of Klebsiella, Serratia and Pseudomonas aeruginosa.     

Bacterial Plasmids in Antarctic Natural Microbial Assemblages

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.     

Conjugal transferability of multiple resistance in Shigella strains

Shigella strains isolated in Japan between 1971 and 1979 were surveyed for drug resistance and distribution of R plasmids. Of 2,510 strains, 89.3% were resistant to either one or various combinations of four drugs, tetracycline, chloramphenicol, streptomycin, and sulfanilamide. About 66% of the Shigella isolates were quadruply resistant. The frequency of isolation of R plasmids from quadruply resistant Shigella strains was the highest when compared with other strains resistant to various combinations of the four drugs. The conjugal transferability of 204 quadruply resistant strains isolated between 1977 and 1979 was tested by various mixed-culture methods. Among the total strains examined, 70.6% carried transferable resistance when tested by the conventional broth culture method, 90.2% transferred their resistance when, in addition the replica-plating method was used and 97.5% could transfer their resistance when the membrane filter method was also used. Although the remaining five strains could not transfer their resistance by any of the mixed culture methods, the drug resistance of four of the five strains was mobilized by the concomitant presence of F-tet or T-kan plasmid. These results indicate that almost all of the quadruple resistance in Shigella isolates was mediated by plasmid.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak.

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Naturally Occurring Penicillinase Plasmids in Staphylococcus aureus

A series of plasmids harbored by naturally occurring penicillin-resistant strains of Staphylococcus aureus were surveyed with a view toward exploring the variability in plasmid-linked marker patterns. Plasmids were transduced from their natural hosts to either of two plasmid-negative laboratory strains by selection for cadmium resistance, and the transductants were tested for all other markers previously found to be plasmid-linked. All of the strains that were able to serve as genetic donors to one of the two stock strains could donate cadmium and lead resistance as linked, plasmid-borne markers. Among the other plasmid markers, a wide variety of patterns was found, including four plasmids that did not carry the penicillinase determinant. Each of the 26 plasmids studied, including the latter 4, was found to belong to one of the two incompatibility sets of penicillinase plasmids previously identified. With the exception of the penicillinase-negative plasmids, which were found in both sets, all the plasmids of incompatibility set I directed the production of penicillinase type A; those belonging to set II directed either type A or type C. Those of set II without exception increased the sensitivity of their host strains to bismuth ion; those of set I carried determinants of bismuth resistance or did not affect the sensitivity of their host to this ion. No other perfect correlations between markers were encountered; in particular, there was no correlation between penicillinase serotype and the excretion of the enzyme. This finding allows the prediction that there is, in addition to all of the markers thus far identified, a plasmid-linked determinant of penicillinase excretion.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

Incidence of antibiotic and metal resistance and plasmid Carriage in Aeromonas isolated from brown bullhead (latalurus nebulosus)

G.W. PETTIBONE, J.P. MEAR AND B.M. SAMPSELL. 1996. Seventy-four strains of Aeromonas were isolated from skin, intestine, kidney and liver of 16 brown bullhead ( Ictalurus nebulosus ). All strains demonstrated multiple antibiotic resistance (MAR) with most strains resistant to rifampin (97%), novobiocin (96%) and vancomycin (85%). The minimum inhibitory concentration of eight metals to selected strains of Aeromonas revealed an apparent toxicity of chromium > copper > cadmium > nickel > mercury > zinc > cobalt > lead, based on the percentage of isolates sensitive to each concentration of metal. Plasmid DNA was found in 36% (27) of the isolates and most plasmid-containing strains had multiple plasmids less than 12 kilobase pairs (kbp) in size. No relationship between plasmid content and antibiotic or metal resistance was found. Plasmid incidence in bacteria isolated from five fish indicated that plasmids were more likely to occur in strains isolated from kidney and liver than in strains isolated from skin and intestine.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

Drug resistance and R plasmids in Pasteurella multocida isolates from swine

A total of 163 strains of Pasteurella multocida isolated from swine were examined for drug resistance and R plasmids. Strains resistant to sulfadimethoxine (Sar), ampicillin (Apr), streptomycin (Smr), kanamycin (Kmr), and chloramphenicol (Cpr) were found in 93.9, 1.8, 16.6, 1.2, and 10.4%, respectively. There were two patterns of drug resistance (Sar and SarCpr) in isolates from nasal cavities, and five patterns (Sar, SarSmr, SarSmrCpr, SarSmrApr, and SarSmrKmrCpr) in isolates from pneumonic lung specimens. Two isolates studied were proved to carry a nonconjugative R plasmid pJY2 or pJY8 with other unidentified plasmids, respectively. pJY2 (3.6 megadaltons) encoding resistance to SarSmr had one cleavage site for EcoRI or HindIII endonuclease and two sites for PstI endonuclease. pJY8 (5.5 megadaltons) encoding resistance to Sar SmrKmrCpr had one EcoRI site and two PstI sites.     

Comparison of virulence factors and R plasmids of Salmonella spp. isolated from healthy and ill swine.

The antibiotic resistance and virulence profiles of Salmonella spp. isolated from healthy (group 1) and ill (group 2) swine were compared. Parameters studied included colicin and siderophore production; mannose-sensitive hemagglutination of erythrocytes; resistance to the lethal effect of serum complement; resistance to antibiotics; and the transmissibility of these characteristics to recipient organisms. Group 1 (19 isolates) had 14 serotypes, and group 2 (20 isolates) had 2 serotypes. Isolates from group 2 were resistant to more antibiotics and had a greater ability to hemagglutinate erythrocytes and transfer R plasmids to recipient organisms, but a lesser ability to produce siderophore than group 1. All 39 isolates resisted the lethal effects of serum complement. Colicin was produced by 1 of 19 from group 1 and 0 of 20 from group 2. A donor Escherichia coli isolated from a pig with enteritis transferred R plasmids to 62% of group 1 and 0% of group 2 Salmonella spp. when they were used as recipient organisms. A transconjugant from the mating of donor E. coli to a group 1 Salmonella spp. was further able to pass an R plasmid to recipient E. coli and salmonellae. Plasmid isolation from group 1 yielded 1 of 19 strains with a 56-megadalton plasmid, while 20 of 20 strains from group 2 contained three to five plasmids from 2.4 to 60 megadaltons in size.     

Comparison of virulence factors and R plasmids of Salmonella spp. isolated from healthy and ill swine

The antibiotic resistance and virulence profiles of Salmonella spp. isolated from healthy (group 1) and ill (group 2) swine were compared. Parameters studied included colicin and siderophore production; mannose-sensitive hemagglutination of erythrocytes; resistance to the lethal effect of serum complement; resistance to antibiotics; and the transmissibility of these characteristics to recipient organisms. Group 1 (19 isolates) had 14 serotypes, and group 2 (20 isolates) had 2 serotypes. Isolates from group 2 were resistant to more antibiotics and had a greater ability to hemagglutinate erythrocytes and transfer R plasmids to recipient organisms, but a lesser ability to produce siderophore than group 1. All 39 isolates resisted the lethal effects of serum complement. Colicin was produced by 1 of 19 from group 1 and 0 of 20 from group 2. A donor Escherichia coli isolated from a pig with enteritis transferred R plasmids to 62% of group 1 and 0% of group 2 Salmonella spp. when they were used as recipient organisms. A transconjugant from the mating of donor E. coli to a group 1 Salmonella spp. was further able to pass an R plasmid to recipient E. coli and salmonellae. Plasmid isolation from group 1 yielded 1 of 19 strains with a 56-megadalton plasmid, while 20 of 20 strains from group 2 contained three to five plasmids from 2.4 to 60 megadaltons in size.     

江苏部分地区食源性和人源沙门氏菌的多重耐药性研究

从江苏省部分地区收集了117个沙门氏菌分离株,其中食物源和人源菌株分别有81株和36株。16种抗生素敏感性试验表明,有111个分离株对2种或2种以上的抗生素有耐药性,人源沙门氏菌分离株的抗生素耐药率比食物源的高,单一抗生素以链霉素耐药率(92.3%,108/117)最高。对5种或5种以上抗生素耐药的分离株有59株(50.4%),其中对特定六种抗生素:氨苄青霉素、氯霉素、链霉素、磺胺、四环素和卡那霉素耐药(ACSSuTK,R型)的菌株有12株。设计18对耐药基因和I类整合子保守区的引物,对36株有不同来源和耐药特征的多重耐药菌株进行耐药基因和I类整合子的检测,PCR扩增结果与抗生素敏感性表型一致。有30株细菌携带有I类整合子,大小为0.3、0.6、1.0、1.2和1.6kb,其中1.6kb(aadA5-dfr17)大小的整合子在25株细菌中分布(24/36)。接合试验表明,氨苄青霉素、氯霉素、链霉素、甲氧苄氨嘧啶和四环素的耐药特性是由接合性质粒携带。结果显示,耐药基因多数由I类整合子和质粒携带,可以通过接合试验发生转移,可移动的DNA成分可能在耐药特性的转移和分布中起到重要作用。     

Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.     

Antibiotic resistance and R-plasmids in food chain Salmonella: evidence of plasmid relatedness.

A large number of strains (1,783) belonging to 15 Salmonella serovars isolated, in Canada, from the three major links of the human food chain were screened for multiple antibiotic resistance and the presence of R-plasmids. Multiresistant strains occurred among animal feed, livestock, and human isolates at frequencies of 4, 22, and 14%, respectively. Conjugation analysis revealed that 58% of the isolates from feeds, 87% of those from livestock, and 89% of the human strains carried all or part of their resistance determinants extrachromosomally on R-plasmids. Conjugative plasmids representing nine different incompatibility groups were detected, with the Inc I alpha group being predominant. Within the limits of the parameters measured, certain of these plasmids show a degree of relatedness suggestive of a common ancestry.     

Incompatibility Reactions of R Plasmids Isolated from Escherichia coli of Animal Origin

The incompatibility reactions of a group of 90 R plasmids, isolated from the fecal Escherichia coli of calves, pigs, and chickens, have been determined against reference plasmids of the incompatibility groups F(11), I, N, P, and W. Twenty plasmids belonged to incompatibility groups F(11), I, N, or P. Forty-nine plasmids were compatible with all of the five reference plasmids. Eight plasmids exhibited marked incompatibility with representatives of two or more of the above groups, whereas two strains showed incompatibility reactions consistent with the coexistence of two compatible plasmids. In addition, 19 plasmids remain untyped because they showed intermediate reactions with one or more reference plasmids.     

Cadmium- and mercury-resistant Bacillus strains from a salt marsh and from Boston Harbor

Bacteria resistant to cadmium or mercury or both were isolated from the Great Sippewissett Marsh (Cape Cod, Mass.) and from Boston Harbor. Many of these metal-resistant isolates were gram-positive aerobic sporeformers, although not necessarily isolated as spores. Although several of the isolated strains bore plasmids, cadmium and mercury resistances appeared to be, for the most part, chromosomally encoded. DNA sequence homology of the gram-positive cadmium- and mercury-resistant isolates was not demonstrable with metal resistance genes from plasmids of either gram-positive (pI258) or gram-negative (pDB7) origin. Cadmium resistance of all the marsh isolates tested resulted from reduced Cd2+ transport. On the other hand, three cadmium-resistant harbor isolates displayed considerable influx but no efflux of Cd2+. Hg-resistant strains detoxified mercury by transforming Hg2+ to volatile Hg0 via mercuric reductase.