Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Drug resistance of Escherichia coli isolated in Nepal.

We collected Escherichia coli strains from 59 Nepalese porters in 1971 and surveyed for their drug resistance. Drug-resistant E. coli strains were isolated from four porters. (TC. CM. SM. SA. APC.)-resistant strains were isolated from two porters and SA- or APC-resistant strains were isolated from each of the others. The R factors were demonstrated from the multiple-resistant E. coli strains.     

Detection of R+ Bacteria in Cultured Marine Fish,Yellowtails (Seriola quinqueradiata)

Many drug-resistant gram-negative bacilli were isolated from the intestinal tracts of yellowtails (Seriola quinqueradiata) cultured on farms in various parts of Kochi and Ehime Prefectures, Shikoku Island. They were Vibrio, Pseudomonas, Proteus, Citrobacter and some unidentified Enterobacteriaceae. Of these drug-resistant strains, Vibrio and Pseudomonas were found to carry R factors in high frequencies. These R factors had four types of resistance markers, SA, SA. CM, SM. CM. ABP. and SA. SM. CM. TC. All R factors were found to belong to the fi^– type. In contrast, only one drug-resistant gram-negative bacillus was detected in a cultured yellowtail on farms near Numazu City, Shizuoka Prefecture.     

Studies of Drug Resistance and R Factors in Bacteria from Pond-Cultured Salmonids

Considerable fractions of gram-negative bacilli, Aeromonas liquefaciens, A. salmonicida, Pseudomonas, Hafnia and unidentified Enterobacteriaceae, isolated from the intestinal tracts of cultured Amago (Oncorhynchus rhodurus macrostomus) from 23 Amago-ponds and of Yamame (O. masou ishikawae) from one pond as well as the water of 3 ponds in Shiga, Gifu and Nagano Prefectures and Tokyo Metropolis in Japan were found to be multiple-drug-resistant. Of these drug-resistant strains, A. salmonicida and A. liquefaciens carried R factors at high frequencies and to be prevalent in many Amago ponds throughout Japan. These R factors had markers of resistance to SA, SA.TC, SA.SM.CM. All the R factors belonged to fi^– type.     

Drug resistance and R plasmids in Escherichia coli strains isolated from broilers

In 1978, 1,021 Escherichia coli strains were isolated from 105 field broilers (F) and 1,058 strains from 106 broilers in a zootechnical experiment station (Z), and their drug-resistance patterns and the presence of conjugative R plasmids were compared. The resistance markers examined were tetracycline (TC), chloramphenicol (CM), streptomycin (SM), sulfonamides (SA), kanamycin (KM), and ampicillin (APC). The populations of individuals that excreted resistant strains were 100% in F and 58% in Z. Frequencies of isolation of drug-resistant strains among the total isolates were 93% in F and 36% in Z, indicating that the resistant strains are a rather high proportion of the intestinal flora in F but are slightly less prevalent in Z. The resistance pattern to (TC.SM.SA.KM) was seen at the highest frequency in both groups. Conjugative R plasmids were demonstrated more frequently in field broilers (F). The results reflect the wide use of antibiotics in the livestock industry, resulting in the appearance of drug-resistant strains mostly due to the presence of R plasmids.     

R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

Immunoprophylactic effects of shiitake mushroom (Lentinula edodes) against Bordetella bronchiseptica in mice

Antimicrobials are used as feed additives to improve growth performance and to prevent subclinical disease challenge in industrial animals. However, these drugs can lead to the development of resistant strains of bacteria. Shiitake mushrooms (SM) (Lentinula edodes) have long been popular as a health food in East Asia. Moreover, SM-derived polysaccharides are well-known as immunostimulants that possess antimicrobial properties. The aim of the present study was to evaluate the immunoprophylactic effects of SM against Bordetella bronchiseptica infection in mice as an initial step towards the development of eco-friendly feed additives to reduce the use of antimicrobials. Although SM had no effect on body weight gain under the un-infected conditions, SM alleviated progressive weight loss and helped in the recovery of body weight in B. bronchiseptica infected mice. Dietary supplementation with SM reinforced bacterial clearance in the infected mice. Of note, SM markedly increased the percentage of various T lymphocytes and the relative mRNA expression levels of tumor necrosis factor-α and interferon-γ in the bronchial lymph node early in the infection. Taken together, these findings suggest that SM could help in the improvement of body weight gain during B. bronchiseptica infection and may enhance the protective immune activity against a subclinical disease challenge, such as B. bronchiseptica infection in mice, probably by a strong stimulation of non-specific immune responses. Hence, SM may provide an alternative to reduce use of antimicrobials. Confirmation of the beneficial effects of SM as a feed additive is now required in industrial animals.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

Formation of the Recombinant between Ampicillin Re istance Determinant and Transfer Factor

In vitro-developed resistant mutants were obtained by inoculating a clinical isolate of Aerobacter cloacae on plates containing various concentrations of ampicillin (APC). This resistance was paralleled by an increase in the formation of β-lactamase. When A. cloacae carrying the nontransmissible APC resistance-determinant (amp) was infected with a T-tet factor, a recombinant T-tet.amp factor was formed; the recombinant being conjugally transmissible and capable of conferring APC resistance on their host. This fact implies the origin of the R factor; the R factor being formed by the recombination of sex factors with nontransmissible drug resistance-determinants.     

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.     

Antimicrobial resistance and presence of <Emphasis Type="Italic">ileS-2</Emphasis> gene encoding mupirocin resistance in clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> sp.

Seventy-eight staphylococcal strains were isolated from surgical-site, blood-stream and other hospital-acquired infections. Eighteen isolates were determined as methicillin (MET)-resistant S. aureus (MRSA), while the remaining were MET-resistant coagulase-negative staphylococci (CoNS). Fifty percent of CoNS strains were multiresistant, while 10 % of isolates were resistant only to β-lactams. Clinical isolates of CoNS were generally more resistant to antimicrobial agents than S. aureus strains. Thirty-nine % of S. aureus strains were resistant only to β-lactams. None of the MRSA strains carried ileS-2 gene; this gene was found in two strains of S. epidermidis.     

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer. Received: 28 January 1997 / Accepted: 3 June 1997     

Drug Resistance of Enteric Bacteria

The strains of gram-negative rod bacteria which are resistant to α-aminobenzylpenicillin and do not harbor the R factors were selected from our stock cultures of clinical origin. It was found that all strains produced β-lactamases which are species-specific in their substrate profiles and classified into three groups; 1) Typical cephalosporinase in the strains of Escherichia freundii, Aerobacter aerogenes, Arizona, Proteus morganii, Proteus rettgeri, Proteus inconstans and a strain GN633 of the Serratia group. 2) Cephalosporinase in the strains of Proteus vulgaris and a strain GN629 of the Serratia group, which has a property of penicillinase to some extent. 3) Penicillinase in the strains of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. It was found that cephalosporinase was generally inducible enzyme, penicillinase was constitutive, and the penicillinase synthesized by the strains carrying R factors belonged to the third group. Penicillinases of two R factors, RGN14 and RGN238 which were isolated in this laboratory and belonged to the penicillinase of the third group, were studied by comparing their substrate profiles and immunological properties. It was demonstrated that penicillinases of RGN14 and RGN238 differed each other, while the penicillinase of K. pneumoniae was quite similar to that of RGN14 both enzymologically and immunologically.     

Frequency of resistance to tetracycline and chloramphenicol amongSalmonella strains isolated in the Netherlands in 1962

Out of 12,472 strains ofSalmonella isolated in the Netherlands in 1962, 1365, or 10.94%, were found to be resistant to tetracycline or chloramphenicol or to both. Compared with the findings of the preceding years (1958/59:2.08%, 1960:1.29%, 1961:3.96%) this is a considerable increase. Of these 1365 strains, 1285, or 94.1%, were resistant to tetracycline and 46, or 3.4%, were resistant to chloramphenicol. The remaining 34 strains, or 2.5%, were resistant to both drugs.Among allSalmonella strains isolated in 1962, 5517 belonged to the speciesS. typhi murium. Of these, 1203 or 21.8%, were resistant to tetracycline. The resistance rates of strains originating from human patients, calves, pigs, other animals and other materials were 24.4%, 37.1%, 15.0%, 8.0% and 5.7% respectively.Factors which may possibly have contributed to the greatly increased frequency of drug resistance inS. typhi murium are: (1) the rapid spread of the use of tetracycline for therapeutic, prophylactic and nutritive purposes, and (2) the possibility of an episome-mediated transfer of drug resistance from relatively harmless intestinal bacteria, such asE. coli, toS. typhi murium.     

Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Imported Pet Birds

Drug resistance in Escherichia coli strains isolated from pet birds (mynahs, macaws, finches, common bengals, parrots, and flamingos) imported into Japan from 10 foreign countries in 1977 and 1978 was investigated. Of the 309 strains isolated from 127 pet birds in the Animal Quarantine Service, 232 (75.1%) were drug resistant. Furthermore, strains resistant to oxytetracycline hydrochloride, dihydrostreptomycin, and sulfadimethoxine were relatively common. Resistance patterns varied from single to sextuple resistance, and 148 (63.8%) of the resistant strains had conjugative R plasmids. These results suggest that the high incidence of drug resistance and R plasmids in E. coli strains isolated from these pet birds may be a reflection of the prophylactic use of antibiotics for the prevention of diseases which increasingly occur with importation of the birds. Furthermore, the results suggest that the birds may be potential reservoirs of drug-resistant E. coli for families who raise and have intimate contact with such birds.     

Recombination Between a Thermosensitive Kanamycin Resistance Factor and a Nonthermosensitive Multiple-Drug Resistance Factor

The thermosensitive kanamycin (KM) resistance factor, R(KM)(t), and a nonthermosensitive multiple-drug resistance factor, R(100), were simultaneously introduced into Escherichia coli and Salmonella typhimurium. The temperature sensitivity of both R factors remained unchanged as long as they replicated independently. Under certain conditions, however, a new thermosensitive R factor harboring resistance markers for kanamycin, streptomycin (SM), and sulfanilamide (SA) was obtained by recombination between the R(KM)(t) and R(100) factors. R factors carrying resistance markers for KM and SA, or for SM and SA, were obtained from the recombinant R(KM SA SM)(t) by spontaneous segregation. Though the R(100) factor has been known as an fi(+) (positive for F-mediated fertility inhibition of its host) type and it does not restrict any coexisting phages, the thermosensitive recombinants of R(100) with R(KM)(t) and their segregants were found to be fi(-) and to restrict the replication of all T-even phages, as does the R(KM)(t) factor. Double infection immunity was not observed between the R(KM)(t) and R(100) factors.     

Effects of Chemotherapeutics on Bacterial Ecology in the Water of Ponds and the Intestinal Tracts of Cultured Fish,Ayu (Plecoglossus altivelis)

Drug-resistant gram-negative bacilli conferred with R factors were isolated with high frequencies from the intestinal tracts of ayu (Plecoglossus altivelis) cultured in ponds, in which chemotherapeutics had often been used, and with relatively low frequencies from ayu which received no administration of chemotherapeutics. Drug-resistant bacteria were also isolated at low frequencies from the intestinal tracts of wild ayu in rivers, as well as from the water of ayu-culturing ponds and some of them carried R factors. The drug-resistant bacteria carrying R factors were Aeromonas liquefaciens, Citrobacter, Enterobacter cloacae, Escherichia coli, Hafnia and unidentified strains. All the R factors were classified as the Fi^–(F) type, except the two R factors detected in an E. coli strain and in an unidentified strain.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Transferable Drug Resistance Among Enterobacteriaceae Isolated from Cases of Neonatal Diarrhea in Calves and Piglets

Fecal specimens were collected on 22 different Nebraska ranches and at the Department of Veterinary Science from young calves and pigs with neonatal diarrhea. Enterobacteriaceae isolated from these fecal specimens were screened for resistance to tetracycline, streptomycin, sulfamethizole, kanamycin, chloramphenicol, colistin, nitrofurantoin, and nalidixic acid. Of the 92 strains studied, 57 were resistant to one or more of these antimicrobial agents. Resistant strains were obtained from all herds involved in the study. The two most common resistance patterns were tetracycline streptomycin sulfamethizole (22 of 57) and tetracycline (13 of 57). None of the strains were resistant to chloramphenicol, colistin, nitrofurantoin, or nalidixic acid. The 57 resistant strains were studied to determine whether the resistance was transferable. Forty-three of the 57 resistant strains could transfer part or all of their resistance pattern to a drug-sensitive recipient. The 43 R(+) strains were obtained from 17 of the 23 herds studied. Considerable variation was observed between different R(+) strains in the frequency of transfer of resistance to a particular drug. In addition, variation in the frequency of transfer of different resistance determinants in individual R(+) strains was noted.