Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.     

Use of genetic and molecular characteristics of R plasmids as an epidemiological marker in an outbreak of hospital infection

Antibiotic sensitivity of 38 strains of enteric bacteria, such as Serratia marcescens Klebsiella pneumoniae and others and Ps. aeruginosa isolated during an outbreak of meningitis in a premature infant resuscitation department was studied. It was shown that all the isolates were multiple resistant, most frequently to 7 antibiotics. All the resistance markers were transferred on conjugation, segregation of some markers being observed. Investigation of the plasmid composition of the clinical strains and transconjugants of E. coli revaled the presence of 2 plasmids with the molecular weights of 40 and 60 Md or one of them. The restriction analysis demonstrated that the plasmids with the same molecular weights isolated from different strains were identical. It was suggested that such plasmids originated from the same source and were distributed by conjugation. The possible part of R plasmids in epidemiological analysis of hospital infections is discussed: the possible part as an additional marker in determination of the infection source and the possible part through its ability to change the host cell phenotype, including the phage and bacteriocin types.     

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.     

Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli.

Plasmid deoxyribonucleic acid (DNA) isolated from each of three antibiotic-resistant clinical strains of Escherichia coli producing the same microcin showed multiple bands upon agarose gel electrophoresis. Transformants selected either for microcin resistance or ampicillin resistance yielded plasmid DNA corresponding in size to only one of the multiple bands. Plasmids, isolated from all three hosts, which determined microcin resistance and microcin production measured about 4 megadaltons by sucrose density, restriction enzyme, and contour length analyses; cleavage of the DNAs by each of eight restriction enzymes showed the same response, and DNA-DNA hybridization indicated complete homology. The antibiotic resistance plasmids of the three host strains were uniformly larger, were of different sizes, and showed different restriction enzyme cleavage patterns. One of these R plasmids (pCP106) also determined the synthesis of the same microcin, and DNA-DNA hybridization studies indicated an approximate 2.4-megadalton homology with the 4-megadalton microcin plasmid pCP101. The microcin plasmids were present at approximately 20 copies per genome equivalent and were nonconjugative, whereas the R plasmids had a copy number of about 1, were conjugative, and could mobilize the microcin plasmid. Microcin plasmid pCP101 showed replication properties similar to those of a number of small multicopy plasmids such as ColE1.     

Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species.

The R factors RP1, R68 and R91 were freely transmissible to and from Pseudomonas aeruginosa, Salmonella typhimurium, and various plant pathogenic Erwinia spp. The antibiotic resistance spectrum of R+ Erwinia recipients was similar to those of other bacteria harboring these R factors, but maximum resistance levels differed with each recipient. The sponstaneous elimination of these factors from the Erwinia strains and the ability to transfer multiple antibiotic resistance suggest that these exist as plasmids in these hosts. Several, but not all, RP1-carrying Erwinia strains were sensitive to the RP1 specific phage PRR1. The R factor R18-1 was also transferred from P. aeruginosa to Erwinia spp. R18-1 was unstable in all Erwinia strains. Stable strains were isolated in which R18-1 could not be eliminated by sodium dodecyl sulfate and could not be transferred to other strains.     

Drug resistance and conjugative R plasmids in fecal Escherichia coli strains isolated from healthy younger animals (chickens, piglets, calves) and children

A total of 11,777 Escherichia coli strains were isolated from 90 chickens, 103 piglets, 96 calves, and 104 children in 1979 in Gunma Prefecture and tested for drug resistance and the presence of conjugative R plasmids. The percentages of individuals that excreted drug-resistant strains were: chickens, 100%; piglets, 99%; calves, 100%; and children, 64%. The frequency of isolation of drug-resistant strains among the total isolates was: chickens, 98%; piglets, 93%; calves, 94%; and children, 41%. Frequency of isolation of R plasmids among the strains tested was: chickens, 48%; piglets, 33%; calves, 38%; and children, 10%. Resistance patterns of the strains isolated most frequently among the four groups were tetracycline (TC), sulfonamides (SA) in single resistance, TC.SA in double resistance, TC.streptomycin (SM).SA in triple resistance and TC.SM.SA. kanamycin (KM) in quadruple resistance. R plasmids were isolated frequently from animals (over 33%) but infrequently from children (about 10%). The high frequency of isolation of drug-resistant strains and R plasmids from animals was caused by the heavy use of chemicals in the period of growth of younger animals.     

Properties of Serratia marcescens strains isolated in septic diseases of newborn infants

As the result of the study of blood and liquor samples from 120 newborns, Serratia marcescens was isolated in 21 cases (17.5 %). 8 strains were isolated from the environment of these patients. Almost all strains isolated from both the patients and the environment (with the exception of one environmental strain) belonged to serotype 04. The isolated S. marcescens strains were resistant to penicillin, ampicillin, streptomycin, kanamycin, oxacillin, methicillin, ceporin and moderately sensitive to polymixin. 2 strains from the environment and 9 strains from the patients were mildly sensitive to gentamicin. In one hospital all isolated strains were found to have 2 transmissive R plasmids with the molecular weight 40 and 60 megadaltons. The presence of R plasmids with the same molecular weight in all S. marcescens strains isolated in this hospital, as well as their serological identity, suggest that in all patients infection originated from a common source.     

Antibiotic resistance in clinical strains of Pseudomonas aeruginosa isolated from 1979-1984

The levels and spectra of drug resistance were determined in 530 strains of P. aeruginosa isolated in hospitals of three cities of the USSR within 1979-1984. Their conjugative R plasmids were searched for and distribution of various type resistance determinants in the composition of these plasmids was investigated. The results were compared with the findings of analogous studies on clinical strains of P. aeruginosa isolated within 1976-1979. It was shown that there were a rise in the relative number of the strains resistant to kanamycin and a decrease in the occurrence of the P. aeruginosa strains resistant to streptomycin, tetracycline and sulfanilamides. The frequency of the kanamycin, carbenicillin and gentamicin resistance genes in the composition of the detected conjugative R plasmids increased. Hybridization of 32P-labeled probes containing various type antibiotic resistance determinants with strains of P. aeruginosa ML (PAO) containing conjugative R plasmids was indicative of wide spread of genes determining APH(3')II and APH(3") and determinants of classes A and C in the composition of the studied plasmids.     

Conjugative R plasmids isolated from hospital strains of Pseudomonas aeruginosa]

It was shown that Pseudomonas aeruginosa hospital strains isolated from patients and environment in the Republican Centre of Burns in Tbilisi contained conjugative R plasmids. The plasmids were marked pM15 and pM19, respectively. The plasmid pM15 determined resistance to carbenicillin, kanamycin and tetracycline and plasmid pM19 determined resistance to carbenicillin, kanamycin, tetracycline, chloramphenicol, gentamicin and streptomycin. Plasmid pM15 had a molecular weight of 45.8 MD and seven sites for EcoRI, six sites for HindIII and five sites for Hpa-I-restrictase. This plasmid, as others, belongs to the Inc-P1 incompatibility group.     

Plasmid characteristics of Salmonella strains of various origins

Salmonella antibiotic-resistant strains, isolated from patients with hospital infections and from various environmental objects, showed lower virulence than antibiotic-sensitive strains in experiments on mice infected by intraperitoneal and enteral routes. Salmonella strains, sensitive to antimicrobial preparations, contained 1-2 plasmids, while those with multiple drug resistance contained 3-10 plasmids varying in their molecular weight. All these strains, with the exception of one laboratory strain, carried a plasmid with a molecular weight of about 60 Md. A decrease in the virulence of Salmonella strains, carrying R-plasmid, with respect to mice, their natural host, in experimental infection by the above-mentioned routes was probably unrelated to the loss of this plasmid. 80% of Salmonella strains with multiple resistance to antibiotics yielded positive results in the keratoconjunctival and conjunctival tests as compared with 42% of sensitive strains. These data suggest that Salmonella strains, carrying R-plasmid, retained pronounced capacity for local colonization.     

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.     

Conjugative R-plasmid resistance of the causative agents of Yersinia infection

Nature, structure, occurrence and drug resistance of 160 strains of Y. pseudotuberculosis and 60 strains of Y. enterocolitica isolated from various sources within 1986-1988 were studied. In the strains of Y. pseudotuberculosis, the cell composition with respect to the requirements in calcium ions as well as the plasmid profiles with determination of the molecular weights of the plasmids in the antibiotic sensitive and resistant pathogens and R(+)-transconjugants were investigated. Some molecular genetic properties of the Yersinia R plasmids were also investigated. Antibiotic polyresistant strains of Y. enterocolitica were the most frequent donors of the R plasmids while the strains of Y. pseudotuberculosis were less frequently the donors, in the resistance pattern of which there were more frequent streptomycin and tetracycline resistance determinants. The conjugative R plasmids of Y. pseudotuberculosis were characterized by strict control of replication, repressed frequency of transfers, and a molecular weight of about 47 MD. Their replicones as a rule contained streptomycin and tetracycline markers determining resistance to streptomycin and tetracycline at the levels of 1250 and 156 micrograms/ml, respectively.     

Drug Resistance and R Factors in the Bowel Bacteria of London Patients before and after Admission to Hospital

The content of drug-resistant coliform bacteria in faecal specimens collected before admission from patients awaiting non-urgent surgery were compared with specimens collected in hospital. Resistant strains of Escherichia coli were isolated from 52% of preadmission specimens and were present in large numbers in 28%. Tetracycline, sulphonamide, and streptomycin resistance were commonest: 60% of resistant strains carried transmissible R factors and multiple resistance was commoner than single. No characteristically resistant intestinal bacteria of any genera were found in hospital specimens as compared with those from outside.     

Localization of genes conferring resistance to selected groups of antibiotics in methicillin-resistant strains of Staphylococcus aureus

Localization of genes conferring resistance to MLS, tetracyclines, chloramphenicol, gentamycin and neomycin in 80 MRSA strains isolated from hospital specimens was determined. The obtained results were compared to DNA patterns of the examined strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that genes of resistance to MLS (ErmI+) in the case of 13 strains were located on chromosome and in the case of 37 strains on plasmids (16 strains had ErmI+ and 21 strains had ErmI-). Genes determining resistance to tetracyclines were localised on chromosome in the case of 39 (23 strains possessed TetK, 11 strains had TetM and 5 strains possessed both TetK and TetM determinants) and in the case of 32 strains on plasmids. Chloramphenicol resistance genes were localised on plasmids in all 30 resistant strains. Genes conferring resistance to gentamycin were present in 31 of the investigated strains on chromosome and in two strains on plasmids. Neomycin resistance genes were plasmid in 34 strains. It was shown that the localization of the resistance genes and the PFGE patterns of the investigated strains were highly correlated.     

Influence of plasmids on bacterial susceptibility to the bactericidal activity of sera. I. Characteristic of R plasmids of enteropathogenic strains of Escherichia coli isolated from clinical cases

The 47 enteropathogenic Escherichia coli strains isolated from the cases of infant diarrhoea were characterized from the point of view of antigenic structures and drug resistance. Six R plasmids were obtained from these strains and were tested for protective activity of E. coli K12 W 1485 against lethal action of sera. Out of three sera tested, the investigated plasmids showed the most evident protective activity upon lethal action of neonatal serum. One of the plasmids appeared even more efficient than R100.1.     

The role of R plasmids in the resistance of bacteria in air

The study of Escherichia coli J 53, used as a model, has revealed that some R plasmids isolated from Serratia marcescens and Klebsiella pneumoniae, found to be the cause of the outbreak of hospital infection, ensure, besides multiple drug resistance, also their viability in the air.     

Specific Distribution of R Factors in Serratia marcescens Strains Isolated from Hospital Infections

Serratia marcescens strains from three hospitals in the city of New York were tested for antibiotic susceptibility patterns and the presence of transmissible antibiotic resistance factors. There appears to be a pattern characteristic for each hospital with regard to the sensitivity to nalidixic acid, tetracycline, chloramphenicol, and sulfonamides, whereas the resistance to ampicillin, cephalothin, and streptomycin is similar in the strains isolated from all three hospitals. In one hospital, a single type of R factor was found which transfers resistance to streptomycin, kanamycin, ampicillin, and sulfonamides, whereas strains isolated from a second hospital transfer only ampicillin resistance. No R factors could be detected in multiply resistant Serratia strains isolated in a third hospital. The presence of a single type of R factor probably reflects the relative ecological isolation of S. marcescens and could be useful for epidemiological studies of hospital infections with Serratia.     

大肠埃希菌耐药性监测及耐药质粒的研究

为了解医院内感染大肠埃希菌的耐药情况,探讨其耐药发生、流行及传播机制,对从临床分离的32株大肠埃希菌进行了药敏试验、质粒图谱分析以及质粒接合、转化试验,结果表明大肠埃希菌对氨苄青霉素的耐药率＞88%,对庆大霉素的耐药率＞75%,其中28株大肠埃希菌检出质粒,均含有一条分子量为5.66 Mu的质粒带,是医院内感染大肠埃希菌的流行质粒.质粒的接合、转化试验证实了质粒具有横向传播的特点,是细菌产生耐药的主要原因.     

Diversity of plasmids and transmission of high-level mupirocin mupA resistance gene in Staphylococcus haemolyticus

The coagulase-negative staphylococci are known for their ability to acquire resistance genes, which limits the choice of therapeutic options for the treatment of infections caused by these microorganisms. In this study, the diversity of high-level mupirocin resistance plasmids (Mup(R) ) was investigated in four strains of Staphylococcus haemolyticus belonging to different pulsed-field gel electrophoresis (PFGE) types or subtypes, isolated in a Brazilian hospital. These strains harbor the mupA gene in large plasmids. In addition, the presence of IS257 sequences flanking the mupA gene was also shown. Two isolates belonging to two different PFGE types exhibited a similar polymorphism for a fragment of the mupA gene and the closest proximal flanking copies of the IS257, suggesting horizontal transmission of S. haemolyticus mupirocin resistance plasmids in the environment and a role of this species as a reservoir of the mupA gene.     

Incidence of the H2 group of plasmids in chloramphenicol-sensitive salmonella isolated in 1974 from clinical sources in Ontario.

Chloramphenicol resistance in salmonella obtained from clinical sources in Ontario was previously found to be often mediated by R plasmids of the H2 incompatibility group. In the present study 40 salmonella strains resistant to one or more of kanamycin, streptomycin, sulfonamides, or tetracycline but sensitive to chloramphenicol, were investigated to determine if they contained R plasmids. Self-transmissible plasmids were isolated from 17 of the strains, and 7 of those showed the bacteriophage inhibition and thermosensitive mechanism of transfer characteristic of H2 plasmids. Entry exclusion and incompatibility experiments confiremd their classification. The results demonstrate that in this population of salmonella, R plasmids of the H2 group are prevalent. Experiments with plasmid-specific phages indicate that the plasmids of this sample, which are not in the H2 group, do not belong to any of the F, I, N, P, or W incompatibility groups.