Study of five penicillinase producing Neisseria gonorrhoeae isolated in Italy

Five penicillinase producing Neisseria gonorrhoeae (PPNG) were isolated from urethral specimens of men admitted to the "Santa Chiara" Hospital (Trento, Italy). All strains proved to be resistant to penicillin and ampicillin, and sensitive to cefuroxime, erythromycin, tetracycline, spectinomycin, nalidixic acid and ciprofloxacin. PPNG plasmid profiles showed that four of the isolates carried the 3.2 MDa "Africa" plasmid and one the 4.5 MDa "Asia" plasmid, the two well-known phenotypes reported in the USA and Europe as well as in Asian and African countries. Membrane matings were performed using N. gonorrhoeae carrying the 24.5 MDa conjugative plasmid as donors and E. coli K12 J 53 as recipient. The transfer of beta-lactamic antibiotic resistance was supported by the presence of 4.5 or 3.2 MDa plasmid bands and by beta-lactamase production in the transconjugants. Restriction analysis of Asian and African plasmids is reported.     

杭州市淋病奈瑟菌质粒酶切图谱分析研究

目的 :了解杭州市淋病奈瑟菌质粒携带及质粒谱型分布情况。方法 :采用碱裂解法对门诊 2 0 0 0年 1月～ 2 0 0 1年 10月分离的 2 0 7株淋病奈瑟菌进行了质粒抽提及质粒谱分型研究 ,并对菌株的青霉素耐药现象和耐药性质粒的关系进行探讨。结果 :2 0 7株淋病奈瑟菌中 194株 (93 .7% )检见质粒带 ,其中含一条质粒带的 112株 (5 4.11% ) ,含两条质粒带的 12株 (5 .8% ) ,含三条带的 70株 (3 3 .82 % ) ,尚有 13株(6.2 8% )未检测到质粒。以 E.coli V5 17细菌质粒作分子量标准 ,测得这些分子量分别为 2 .6、4.5、和 2 4.5Md。质粒谱型以 2 .6 4.5 2 4.5 Md(3 3 .82 % )多见。结论 :杭州地区质粒酶切图谱的分析研究有助于淋病的治疗和防治 ,这将对该地区淋病奈瑟菌的分子流行病学调查和淋病监控提供依据     

Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability

Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi^–, and the 44.5 Md Kc^r plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, transfer frequencies, and in vitro stability, were studied. Plasmids generally showed a wide host range and high stability in the transconjugants tested. It could be concluded that these plasmids may be widely disseminated in the environment studied.     

Antibiotic susceptibility of Neisseria gonorrhoeae in relation to serogroups

Susceptibility testings, by means of the agar-dilution method, were performed on nine antibiotics of 138 non-PPNG (beta-lactamase-producing = PPNG) strains, 17 of which originated from Thailand, and 88 PPNG isolates. The gonococcal strains were sero-grouped by the co-agglutination method and classified among the sero-groups W I, W II or W III. Statistically significant differences in antibiotic susceptibility between non-PPNG strains of the three sero-groups could be demonstrated with strains belonging to sero-group W I as the most sensitive and W III isolates as the most resistant. Non-PPNG strains from Thailand were significantly more resistant than the other non-PPNG isolates of the same sero-group. There was, however, no significant difference in resistance between non-PPNG strains from Thailand and PPNG isolates of the same sero-group. PPNG strains of sero-group W I were significantly more sensitive than PPNG strains of sero-group W II, whereas there was no significant difference between PPNG isolates of sero-groups W II and W III. Non-PPNG strains, not from Thailand, of sero-groups W I and W II were significantly more susceptible to non beta-lactam antibiotics than PPNG strains of corresponding sero-groups, while no such difference could be demonstrated for non-PPNG and PPNG isolates of sero-group W III.     

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.     

Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Characterization of plasmids from antibiotic-resistant Shigella isolates by agarose gell electrophoresis.

Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1.0 to 24.5 Mdal were present. Mobilization of the 5.5 Mdal SuSm plasmid and a 1.0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24.5 Mdal Fi- plasmid carrying no known drug resistance determinants.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry

Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.     

The R-factors of multiple antibiotic resistant faecal coliforms isolated from a domestic dog

The antibiotic resistant faecal flora of a domestic dog suffering from an acute enteric infection was examined. The flora exhibited overall resistance to a wide variety of antibiotics. However, following restoration of the animal to normal health, overall resistance to ampicillin (Ap), tetracycline (Tc), chloramphenicol (Cm) and streptomycin (Sm) was lost, although low numbers of bacteria resistant to these four antimicrobial agents could still be isolated up to one year later. A total of 11 strains were purified for further study. All 11 were positively identified as Escherichia coli and shown to be resistant to various combinations of the above antibiotics, and additionally to kanamycin (Km). Each strain harboured from one to five plasmids, although only four proved capable of transferring antibiotic resistance to Escherichia coli K-12. One of the strains was found to harbour two conjugal plasmids pNJ101 (60 Md) and pNJ102 (133 Md) which coded for resistance to Cm, Tc, Ap and Cm, Tc, Km respectively. A third plasmid pNJ103 (29 Md) remains cryptic. The possession of the two plasmids pNJ101 and pNJ102 appears to be an unstable situation as variants arose harbouring one or other of the plasmids.     

Plasmid detection and isolation in strains of Clostridium acetobutylicum and related species

Summary Twenty-one strains of Clostridium acetobutylicum, C. butylicum and Clostridium saccharoperbutylacetonicum were examined. Seven of them contained extrachromosomal DNA molecules, with a size ranging from 2.6 to more than 50 megadaltons. Strain M1 carries a small plasmid of 2.6 megadaltons, AB10 at least one plasmid of 2.6 megadaltons, AB12 one plasmid of 5.2 megadaltons, AB14 and AB16 a plasmid of about 7 megadaltons and a large one of more than 50 megadaltons, AB17 carries at least one plasmid of 6.7 megadaltons and AB18 two plasmids (4–6 megadaltons and 10–12 megadaltons). All of them are cryptic as at present no function can be correlated with their presence in a bacterial strain.     

The ecology of gonococcal plasmids.

Of 261 strains of Neisseria gonorrhoeae examined for plasmids, 6 were plasmid-free, 217 contained only a small multicopy 2.6 X 10(6) dalton plasmid and 38 carried a large 24.5 X 10(6) dalton plasmid. Restriction enzyme digests and DNA-DNA hybridization studies revealed that the large plasmids isolated between 1940 and 1978 share a common core of DNA sequences (70 to 100%) and represent a group of closely related molecules.     

Plasmids of Azospirillum brasilense

The cells from natural isolates of A. Brasilense were found to harbour 1 to 4 plasmids with the molecular masses within the 27-300 Md range. 100 Md plasmids are specific for this bacterial species. Strains isolated from the roots of cereals (wheat, maize, barley) have more heterogeneous plasmid composition as compared to the strains isolated from soil.     

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.