Transferable or mobilisable antibiotic resistance in Shigella dysenteriae types 1, 2, 3, 4, 6 and 7 isolated in Ethiopia during 1974-85

A total of 199 Shigella dysenteriae isolates resistant to one or more antibiotics and belonging to types 1, 2, 3, 4, 6 and 7 was examined by one-step broth mating with Escherichia coli K12 and, if non-conjugative, additionally by triparental crosses with the conjugative plasmids X and delta. Of the S. dysenteriae type 1 (Shiga's bacillus) strains, 96% harboured conjugative plasmids. During 1974-79, isolates of Shiga's bacillus carried conjugative plasmids coding for ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance that transferred at low frequencies (less than 10(-4). After 1980, however, about 50% of isolates of Shiga's bacillus with this resistance (R)-type carried conjugative plasmids that transferred at high frequencies (10 degrees-10(-2)) and that expressed the ACT determinant only. The introduction of a new clone of Shiga's bacillus into Ethiopia in 1980 is suspected. Conjugative plasmids coding for SSuT resistance were detected in S. dysenteriae types 2, 3, and 4. Non-conjugative SSu determinants in S. dysenteriae type 3 were mobilised by conjugative plasmids X and delta. R-type CSSuT in strains of types 2 and 7, and R-type ACST in type-3 strains were neither transferable nor mobilisable and are probably determined chromosomally.     

Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.     

Antibiotic resistance in Staphylococcus aureus isolated at an Australian hospital between 1946 and 1981

A total of 517 strains of Staphylococcus aureus isolated at a hospital in Melbourne, Australia between 1946 and 1981 was examined for resistance to a range of antimicrobial agents and for the presence of plasmid DNA. The use of mixed-culture transfer and restriction endonuclease analysis showed that the determinants for resistance to penicillin and to the heavy metals were carried by several related plasmids of (15-23) X 10(6) mol. wt, and that tetracycline resistance was encoded on a plasmid of 2.8 X 10(6) mol. wt in strains isolated before 1970. These phenotypes were chromosomally encoded in the majority of strains isolated thereafter. Resistance to chloramphenicol throughout the study period was plasmid-mediated. Of five aminoglycoside-resistance phenotypes, one was plasmid-mediated and three were chromosomally encoded. The remaining phenotype, specifying low-level gentamicin resistance, was found to be located on the chromosome of early isolates, but in later strains was borne by an 18 X 10(6) mol. wt plasmid which also encoded resistance to quaternary ammonium compounds.     

Concurrent transfer and recombination between plasmids encoding for heat-stable enterotoxin and drug resistance in porcine enterotoxigenicEscherichia coli

The frequency of and genetic mechanisms for simultaneous transfer of genes encoding for tetracycline and sulpha-streptomycin resistance, heat-labile (LT) and heat-stable (ST-mouse) enterotoxin production in porcine enterotoxigenicEscherichia coli toEscherichia coli K12 were investigated. SevenE.coli strains of 0-group 149 were studied by conjugation and transformation experiments. All strains transferred tetracycline-resistance plasmids at a high frequency. No interaction was observed between these plasmids and those encoding for LT production. However, most tetracycline-resistant recipient cells were ST-mouse⁺ following recombination events between plasmids encoding for colicin B and ST-mouse production and plasmids encoding for tetracycline resistance. Alternatively, when selecting for sulpha or streptomycin resistance a majority of the transconjugants were also ST-mouse⁺, as plasmids coding for sulpha and streptomycin were mobilized by the colicin B and ST-mouse encoding plasmid. Since the simultaneous transfer of genes encoding for drug resistance, colicin B and ST-mouse production are common events in vitro, they might also occur frequently in vivo during antibiotic selective pressure.     

Multidrug resistant Salmonella typhi in asymptomatic typhoid carriers among food handlers in Namakkal district, Tamil Nadu

Purpose: To screen Salmonella typhi in asymptomatic typhoid carriers and to find out drug resistance and ability of the strains to transmit drug resistance to other bacteria Methods: Cultural characters, biochemical tests, antibiotic sensitivity test (disc diffusion), agarose gel electrophoresis, and conjugation protocols were done. Thirty five stool samples were collected from the suspected food handlers for the study. Results: Among 35 samples, (17.14%) yielded a positive result. Out of these 4 (20.0%) were women and 2 (13.33%) were men. The isolates were tested with a number of conventional antibiotics viz, amikacin, amoxicillin, ampicillin, chloramphenicol, ciprofloxacin, co-trimaxazole, rifampicin, gentamicin, nalidixic acid, ofloxacin and tetracycline. Five isolates were having the multidrug resistant character. Four (66.66%) multidrug resistant isolates were found to have plasmids, while one (16.66%) multidrug resistant isolate had no plasmid and the chromosome encoded the resistance. Only one strain (16.66%) showed single antibiotic resistance in the study and had no plasmid DNA. The molecular weights of the plasmids were determined and found to be 120 kb.The mechanism of spreading of drug resistance through conjugation process was analyzed. In the conjugation studies, the isolates having R⁺ factor showed the transfer of drug resistance through conjugation, which was determined by the development of antibiotic resistance in the recipients. Conclusion: This study shows that drug resistant strains are able to transfer genes encoding drug resistance.     

Antibiotic Resistance Patterns and Plasmid Profiles of Salmonella typhimurium Isolates in Turkey

 To understand the resistance mechanisms present in 75 isolates of Salmonella typhimurium derived from clinical infections in Turkey, antimicrobial resistance patterns and associated plasmids were investigated. Among the 22 strains that produced extended-spectrum β-lactamase (ESBL), 20 were resistant to aminoglycosides and 12 to trimethoprim-sulfamethoxazole. Strains that did not produce ESBL did not express aminoglycoside or trimethoprim-sulfamethoxazole resistance, although 27 of them were ampicillin resistant. None of the strains were resistant to imipenem or fluoroquinolones. Nineteen strains producing ESBL carried a plasmid of >100 MDa. Seven ESBL-producing strains conjugally transferred their ESBLs and trimethoprim-sulfamethoxazole resistance. No correlation was found between the resistance patterns and plasmids in non-ESBL-producing strains.     

Prevalence and profiles of plasmids inPseudomonas aeruginosa

The value of plasmid profile determination as an epidemiological tool inPseudomonas aeruginosa infections was investigated by determining the prevalence of plasmids in 450Pseudomonas aeruginosa strains and comparing the technique with other epidemiological tools. Since only 13.9% of these strains harbored plasmids and the majority of these plasmids were antibiotic resistant, the technique appeared to be less appropriate as an epidemiological tool in this organism than other techniques. Comparison of results obtained from plasmid profile determinations with those from antibiotyping, serotyping and pyocin typing in 50 non-epidemic strains showed the technique gave highly reproducible results and was sensitive; its ability to discriminate could be improved by additionally performing conjugation assays and hydrolysis of plasmidic DNA with restriction enzymes. It is concluded that plasmid profiles provide important epidemiological information onPseudomonas aeruginosa infections when performed in conjunction with either serotyping or, more importantly, pyocin typing.     

Plasmids coding for enterotoxins,K88 antigen and colicins in porcineEscherichia coli strains of O-group 149

This study was carried out to determine whether the strong epidemiological correlation observed in Sweden between production of the adhesin K88, the heat-stable (ST) and the heat-labile (LT) enterotoxins inE. coli strains of O-group 149 isolated from piglet diarrhea might be explained by linkage of their genetic determinants. From 22 different isolates plasmids coding for these virulence factors were investigated by conjugation and transduction experiments and analysis on agarose gels. The genes coding for ST production could be transferred by selection for antibiotic resistance, but behaved as transposable elements most often residing on a 55 Mdal plasmid coding for colicin B. The genes coding for raffinose fermentation and K88 antigen production were located on a 45 Mdal plasmid and the genes coding for LT production on plasmids within the 45–70 Mdal size. Thus the epidemiological importance and spread of this O-group in Sweden was explained by its stable content of two or three virulence plasmids, which could be transferred independently of one another.     

Variability of resistance plasmids in coagulase-negative staphylococci and their importance as a reservoir of antimicrobial resistance

Coagulase-negative staphylococci (CoNS) are an important cause of human and animal diseases. Treatment of these diseases is complicated by their common antimicrobial resistance, caused by overuse of antibiotics in hospital and veterinary environment. Therefore, they are assumed to serve as a reservoir of resistance genes often located on plasmids. In this study, we analyzed plasmid content in 62 strains belonging to 10 CoNS species of human and veterinary origin. In 48 (77%) strains analyzed, 107 different plasmids were detected, and only some of them showed similarities with plasmids found previously. In total, seven different antimicrobial-resistance genes carried by plasmids were identified. Five of the CoNS staphylococci carried plasmids identical with either those of other CoNS species tested, or a well characterized Staphylococcus aureus strain COL, suggesting plasmid dissemination through horizontal transfer. To demonstrate the possibility of horizontal transfer, we performed electroporation of four resistance plasmids among Staphylococcus epidermidis, Staphylococcus petrasii, and coagulase-positive S. aureus strains. Plasmids were transferred unchanged, were stably maintained in recipient strains, and expressed resistance genes. Our work demonstrates a great variability of plasmids in human and veterinary staphylococcal strains and their ability to maintain and express resistance plasmids from other staphylococcal species.     

Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine

As part of a longitudinal study of antimicrobial resistance among salmonellae isolated from swine, we studied 484 Salmonella enterica subsp. enterica serovar Typhimurium (including serovar Typhimurium var. Copenhagen) isolates. We found two common pentaresistant phenotypes. The first was resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (the AmCmStSuTe phenotype; 36.2% of all isolates), mainly of the definitive type 104 (DT104) phage type (180 of 187 isolates). The second was resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (the AmKmStSuTe phenotype; 44.6% of all isolates), most commonly of the DT193 phage type (77 of 165 isolates), which represents an unusual resistance pattern for DT193 isolates. We analyzed 64 representative isolates by amplified fragment length polymorphism (AFLP) analysis, which revealed DNA fingerprint similarities that correlated with both resistance patterns and phage types. To investigate the genetic basis for resistance among DT193 isolates, we characterized three AmKmStSuTe pentaresistant strains and one hexaresistant strain, which also expressed resistance to gentamicin (Gm phenotype), all of which had similar DNA fingerprints and all of which were collected during the same sampling. We found that the genes encoding the pentaresistance pattern were different from those from isolates of the DT104 phage type. We also found that all strains encoded all of their resistance genes on plasmids, unlike the chromosomally encoded genes of DT104 isolates, which could be transferred to Escherichia coli via conjugation, but that the plasmid compositions varied among the isolates. Two strains (strains UT08 and UT12) had a single, identical plasmid carrying bla(TEM) (which encodes ampicillin resistance), aphA1-Iab (which encodes kanamycin resistance), strA and strB (which encode streptomycin resistance), class B tetA (which encodes tetracycline resistance), and an unidentified sulfamethoxazole resistance allele. The third pentaresistant strain (strain UT20) was capable of transferring by conjugation two distinct resistance patterns, AmKmStSuTe and KmStSuTe, but the genes were carried on plasmids with slightly different restriction patterns (differing by a single band of 15 kb). The hexaresistant strain (strain UT30) had the same plasmid as strains UT08 and UT12, but it also carried a second plasmid that conferred the AmKmStSuGm phenotype. The second plasmid harbored the gentamicin resistance methylase (grm), which has not previously been reported in food-borne pathogenic bacteria. It also carried the sul1 gene for sulfamethoxazole resistance and a 1-kb class I integron bearing aadA for streptomycin resistance. We also characterized isolates of the DT104 phage type. We found a number of isolates that expressed resistance only to streptomycin and sulfamethoxazole (the StSu phenotype; 8.3% of serovar Typhimurium var. Copenhagen strains) but that had AFLP DNA fingerprints similar or identical to those of strains with genes encoding the typical AmCmStSuTe pentaresistance phenotype of DT104. These atypical StSu DT104 isolates were predominantly cultured from environmental samples and were found to carry only one class I integron of 1.0 kb, in contrast to the typical two integrons (InC and InD) of 1.0 and 1.2 kb, respectively, of the pentaresistant DT104 isolates. Our findings show the widespread existence of multidrug-resistant Salmonella strains and the diversity of multidrug resistance among epidemiologically related strains. The presence of resistance genes on conjugative plasmids and duplicate genes on multiple plasmids could have implications for the spread of resistance factors and for the stability of multidrug resistance among Salmonella serovar Typhimurium isolates.     

Plasmid associated virulence properties of environmental isolates of Aeromonas hydrophila.

The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptible to all the antimicrobial agents tested; the highest incidences of resistance were to tetracycline (96.7%), prystanamycin (93.3%), ampicillin (91.7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4.2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultaneously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60% of the strains simultaneously possessed plasmids and haemolytic activity.     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.     

Molecular and phenotypical investigation of ciprofloxacin resistance among Campylobacter jejuni strains of human origin: high prevalence of resistance in Turkey

Campylobacteriosis is one of the most frequently reported zoonoses worldwide. The well-documented increase in the ciprofloxacin resistance has increased the importance of rapid detection of the resistance. The incidence of ciprofloxacin resistance was investigated using real-time PCR. Identification of one hundred and fifty-eight strains was performed by PCR. Minimum inhibitory concentration (MIC) of ciprofloxacin was determined by Epsilometer test. Following the confirmation of the efficiencies of singleplex real-time PCR methods using two different probes, a cytosine to thymine point mutation at codon 86 was detected by allelic discrimination. Of the 158 strains, 114 (72.2%) were determined to be resistant to ciprofloxacin. The MIC₅₀ and the MIC₉₀ of ciprofloxacin were found to be 8 and ≥32 mg/L, respectively. By real-time PCR, the presence of the mutation was confirmed in all, but one, resistant strains and the absence of the mutation was demonstrated in all, but one, susceptible strains. The rate of resistance is high among C. jejuni strains and ciprofloxacin should not be used in the treatment of such infections in Turkey. A cytosine to thymine mutation is the most frequently detected mechanism for the resistance. Real-time PCR can be used for the quick screening of the resistance.     

Transfer of Rhizobium leguminosarum Sym plasmids to R. meliloti and stability of resident and transferred plasmids

The recombinant symbiotic (Sym) plasmids pIJ1008 and pIJ1019 and the natural Sym plasmid pJB5JI of Rhizobium leguminosarum were transferred to derivatives of the Rhizobium meliloti strain Rm41 and to local wild strains of R. meliloti to investigate their transfer frequency and stability. Transfer to the latter strains was only successful through two-step conjugations, i.e. transfer to a compatible R. meliloti strain followed by transfer to wild strains. The transferred Sym plasmids and the resident plasmids of R. meliloti with a molecular weight up to approximately 300 Md showed instability, resulting in the elimination of the latter, whereas the R. meliloti megaplasmids and their symbiotic properties were maintained. The natural plasmid pJB5JI was stable in free living transconjugants as well as those in symbiosis with alfalfa or pea. The two recombinant Sym plasmids showed deletions or structural changes dependending on the genotype of the recipient and the transferred plasmids. During symbiosis these plasmids may be further deleted or completely eliminated. The consequences for gene transfer programs are discussed in the paper.     

Haemophilus influenzae: comparison of respiratory tract isolates with genitourinary tract isolates.

Haemophilus influenzae isolates recovered from the genitourinary (GU) tract were shown to have a significantly different biotype distribution compared with respiratory tract isolates. Biotype IV strains were recovered more commonly from the GU tract, and most strains were non-serotypable. Antibiotic-susceptible strains isolated from the GU tract more frequently harbored plasmids of less than 10 megadaltons than did antibiotic-susceptible respiratory tract strains. One 2.8-megadalton plasmid resident in a GU tract isolate and one 1.8-megadalton plasmid resident in a respiratory tract isolate were shown to be related to the small ampicillin resistance plasmids previously described in H. influenzae, Haemophilus parainfluenzae, Haemophilus ducreyi, and Neisseria gonorrhoeae. This supports the suggestion that these ampicillin resistance plasmids originated by transposition or recombination of the ampicillin transposon (TnA) with cryptic endogenous Haemophilus plasmids.     

Restriktion und Modifikation bei Staphylococcus aureus: Eigenschaften von Resistenzplasmiden und Prophagen

Experiments on elimination and transfer of resistance-plasmids in S. aureus (controlling resistance to penicillin, chloramphenicol and oxytetracycline) show that these plasmids have no restricting influence on phages used for typing of staphylococci. Prophages in lysogenic strains control a mechanism of restriction and modification which is active on phages and on chromosomal markers. The resistance-plasmids used in these experiments are insensitive to prophage controlled restriction.     

Heavy metal resistant Arthrobacter sp. — A tool for studying conjugational plasmid transfer between Gram-negative and Gram-positive bacteria

The role of two heavy metal-resistant strains of the Gram-positive genus Arthrobacter sp. as a tool in studying conjugational plasmid transfer between Gram-positive and Gram-negative bacteria is described. The high nickel resistance and the cobalt resistance of Arthrobacter sp. strain RM1/6 could be transferred to Arthrobacter sp. strain WS14. IncQ plasmids (pKT240, pKT240::czc, pML10) could be mobilized from E. coli into Arthrobacter spp. strains; antibiotic (Km, Ap, Tc)¹) and heavy metal (Co) resistance genes were expressed in the recipient strains. IncQ plasmid pKT240 could be mobilized between Arthrobacter spp. strains. IncP plasmid RP4::Tn4371 was transferred from A. eutrophus to Arthrobacter sp., RP4-mediated antibiotic resistance to Km was expressed in the recipient strain.     

Molecular Characterization and Transfer Among Staphylococcus Strains of a Plasmid Conferring High-Level Resistance to Mupirocin

 In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42  °C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0×10^–9/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcus epidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcus haemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.     

Co-transfer of plasmids in association with conjugative transfer of mupirocin or mupirocin and penicillin resistance in methicillin-resistant Staphylococcus aureus

Two distinct strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients in a dermatology ward were also resistant to mupirocin. The mupirocin resistance plasmids from both strains were indistinguishable by EcoRI and HindIII restriction digest analysis, except for the presence of genes apparently mediating penicillinase production in some transconjugants. Conjugative transfer of the plasmid mediating mupirocin resistance from one of these strains to a recipient S. aureus was accompanied in some cases by co-transfer of plasmids mediating resistance to tetracycline or erythromycin; in some instances a plasmid which possessed no apparent resistance markers was also transferred. The second strain demonstrated conjugative transfer of penicillin and mupirocin resistance as well as transfer of a plasmid mediating gentamicin resistance, but transfer of erythromycin resistance was not apparently plasmid-mediated.     

Azithromycin resistance inCampylobacter jejuni andCampylobacter coli

The MICs of erythromycin, azithromycin and ciprofloxacin were determined for 60 human fecal isolates ofCampylobacter. Of these, 30 strains selected on the basis of their resistance to erythromycin by disk diffusion were highly resistant to both erythromycin and azithromycin. Nine of these selected isolates were resistant to ciprofloxacin. The remaining 30 strains were non-selected, consecutive isolates ofCampylobacter susceptible to erythromycin by disk diffusion and were shown to be two- to five-fold more susceptible to azithromycin than to erythromycin as determined by MIC testing.