Apramycin and gentamicin resistance in Escherichia coli and salmonellas isolated from farm animals.

Since the aminoglycoside antibiotic apramycin was licensed for veterinary use in 1980, all isolates of Escherichia coli and salmonellas received at the Central Veterinary Laboratory have been monitored for resistance to apramycin and the related antibiotic gentamicin. During the period 1982-4, the incidence of resistance in E. coli to apramycin increased from 0.6% in 1982 to 2.6% in 1984. In salmonellas the incidence of resistance to apramycin increased from 0.1% in 1982 to 1.4% in 1984. Resistance to both apramycin and gentamicin was detected in six different salmonella serotypes, although an isolate of Salmonella thompson from poultry was resistant to gentamicin but not apramycin. Most of the cultures were isolated from pigs, although the incidence of apramycin resistance in S. typhimurium (DT 204C) from calves has shown a recent dramatic increase. All the isolates with one exception produced the enzyme aminoglycoside 3-N-acetyltransferase IV (ACC(3)IV). The resistance was transferable by conjugation in most of the strains examined, and the plasmids specifying the resistance have been found to belong to a number of different incompatibility groups. Plasmids from three E. coli strains were compatible with all the reference plasmids and belonged to a previously undescribed group which was investigated further. It is suggested that bacteria from humans should be examined for resistance to apramycin and gentamicin to determine the possibility of the antibiotic-resistance bacteria, and their genes, spreading from animals to humans.     

Urinary isolates of apramycin-resistant Escherichia coli and Klebsiella pneumoniae from Dublin.

Twenty-two gentamicin-resistant urinary isolates of Escherichia coli and five gentamicin-resistant urinary isolates of Klebsiella pneumoniae from a Dublin hospital were examined for resistance to the veterinary aminoglycoside antibiotic apramycin. Five isolates of E. coli and one isolate of K. pneumoniae were found to be resistant. The apramycin-resistant isolates, which were also resistant to the veterinary anthelmintic agent hygromycin B, hybridized with a DNA probe for the gene encoding the enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC(3)IV). Resistance to apramycin and hygromycin B was co-transferable in four of the five isolates of E. coli and the isolate of K. pneumoniae. In one isolate of E. coli apramycin resistance was not transferable. On the basis of their restriction enzyme digestion profiles and the antimicrobial resistance traits encoded, the transferable plasmids encoding resistance to apramycin and hygromycin B comprised three distinct types. Genetic linkage between the gene encoding AAC(3)IV and genes encoding resistance to ampicillin and either tetracycline or trimethoprim, means that the relatively widespread use of these antimicrobial agents provides a selective pressure for the persistence of resistance to apramycin and gentamicin even in the absence of bacterial exposure to aminoglycosides.     

Human isolates of apramycin-resistant Escherichia coli which contain the genes for the AAC(3)IV enzyme.

Gentamicin-resistant Escherichia coli isolated at different periods from patients in two hospitals were tested for resistance to the aminoglycoside antibiotic apramycin. Twenty-four of 93 (26%) gentamicin-resistant isolates collected from the Royal Liverpool Hospital between 1981 and 1990 were resistant to apramycin. Thirteen isolates were highly resistant to apramycin (minimal inhibitory concentration (MIC) > or = 1024 micrograms/ml), were also resistant to gentamicin, netilmicin and tobramycin, and hybridized with a DNA probe derived from the aminoglycoside acetyltransferase (3)IV (AAC(3)IV) gene. The proportion of gentamicin-resistant isolates which had high level resistance to apramycin increased from 7% in 1981-5 to 24% in 1986-90. Twelve gentamicin-resistant E. coli from Guy''s and St Thomas''s Hospital isolated between 1977 and 1980 were also tested for resistance to apramycin. For five of these isolates the MICs of apramycin was 32-256 micrograms/ml. None was shown to have a conjugative plasmid carrying resistance to apramycin and only one hybridized with the DNA probe for the AAC(3)IV enzyme.     

Apramycin-resistant Escherichia coli isolated from pigs and a stockman.

Escherichia coli serotype O147:K89:K88a,c was found to be associated with outbreaks of diarrhoea in preweaner pigs of up to 4 weeks of age on a pig unit. Resistance to apramycin, gentamicin, netilmicin, tobramycin and other antibiotics was associated with conjugative plasmids of approximately 62 kb. The presence of a gene which encoded for the aminoglycoside acetyltransferase enzyme AAC(3)IV was confirmed by DNA hybridization. Samples collected during the following 12 months revealed widespread dissemination of these resistance plasmids in non-serotypable, non-haemolytic E. coli throughout the farm. Apramycin-resistant E. coli were also isolated from a stockman and it appeared from plasmid profile analysis and antibiotic sensitivity testing that the human isolates carried the same plasmid as that carried by the porcine E. coli. Klebsiella pneumoniae, with a slightly smaller conjugative plasmid and similar resistance pattern, was isolated from the stockman''s wife.     

Apramycin and gentamicin resistances in indicator and clinical Escherichia coli isolates from farm animals in Korea

A total of 1921 Escherichia coli isolated from healthy animals (501 from cattle, 832 from pigs, and 588 from chickens) and 237 isolates from diseased pigs were tested to determine the prevalence of apramycin and gentamicin resistance in Korea during 2004-2007. Apramycin/gentamicin resistances observed in healthy cattle, pigs, and chicken were 0.2%/0.6%, 11.2%/13.6%, and 0.5%/18.2%, respectively. Gentamicin/apramycin resistance was much higher in E. coli isolated from diseased pigs (71/237, 30.0%) than in those from healthy pigs (93/832, 11.2%). The aminoglycoside resistance gene content of all apramycin-gentamicin-resistant E. coli isolates (n= 164) was determined by polymerase chain reaction. Of seven different types of aminoglycoside resistance genes tested, five kinds were detected in the 164 isolates: aac(3)-IV, aac(3)-II, aac(3)-III, ant(2')-I, and armA. All apramycin-resistant E. coli contained the aac(3)-IV gene. About half of the resistant isolates carried only the aac(3)-IV gene and the other half carried other genes in addition to aac(3)-IV. The results of the present study suggest that humans are at risk of gentamicin resistance from apramycin use in animals.     

Apramycin resistance plasmids in Escherichia coli: possible transfer to Salmonella typhimurium in calves.

An outbreak of salmonellosis in calves was monitored for persistence of Salmonella typhimurium excretion in faeces and the effect of treatment with apramycin. Prior to treatment apramycin-resistant Escherichia coli were present but all S. typhimurium isolates were sensitive. Following the treatment of six calves with apramycin, apramycin-resistant S. typhimurium were isolated from two treated calves and one untreated calf. Plasmid profiles of E. coli and S. typhimurium were compared and plasmids conferring resistance to apramycin and several other antibiotics were transferred by conjugation in vitro from calf E. coli and S. typhimurium isolates to E. coli K-12 and from E. coli to S. typhimurium. The plasmids conjugated with high frequency in vitro from E. coli to S. typhimurium, and hybridized to a DNA probe specific for the gene encoding aminoglycoside acetyltransferase 3-IV (AAC(3)-IV) which confers resistance to apramycin, gentamicin, netilmicin and tobramycin.     

Characterization of plasmids conferring resistance to gentamicin and apramycin in strains of Salmonella typhimurium phage type 204c isolated in Britain

In Salmonella typhimurium phage type 204c isolated in Britain, gentamicin resistance is specified by plasmids of the I1 compatibility group which also confer resistance to apramycin. These plasmids have been subdivided into three types within the I1 group on the basis of their antibiotic resistance specificity, their ability to produce colicin Ib and their restriction enzyme digest fragmentation patterns. All three have been identified in strains from cattle, but as yet only two types have been found in strains from humans. It is suggested that the use of apramycin in animal husbandry is responsible for the appearance of gentamicin resistance in multiresistant strains of phage type 204c, a phage type already epidemic in bovine animals and with an increasing incidence in humans.     

Characterization of plasmids conferring resistance to gentamicin and apramycin in strains of Salmonella typhimurium phage type 204c isolated in Britain.

In Salmonella typhimurium phage type 204c isolated in Britain, gentamicin resistance is specified by plasmids of the I1 compatibility group which also confer resistance to apramycin. These plasmids have been subdivided into three types within the I1 group on the basis of their antibiotic resistance specificity, their ability to produce colicin Ib and their restriction enzyme digest fragmentation patterns. All three have been identified in strains from cattle, but as yet only two types have been found in strains from humans. It is suggested that the use of apramycin in animal husbandry is responsible for the appearance of gentamicin resistance in multiresistant strains of phage type 204c, a phage type already epidemic in bovine animals and with an increasing incidence in humans.     

Effects of antibiotic use in sows on resistance of E. coli and Salmonella enterica Typhimurium in their offspring

To determine effects of exposure of parental animals to antibiotics on antibiotic resistance in bacteria of offspring, sows were either treated or not treated with oxytetracycline prior to farrowing and their pigs were challenged with Salmonella enterica Typhimurium and treated or not treated with oxytetracycline and apramycin. Fecal Escherichia coli were obtained from sows, and E. coli and salmonella were recovered from pigs. Antibiotic resistance patterns of isolates were determined using a minimum inhibitory concentration (MIC) analysis. Polymerase chain reaction (PCR) and electroporation were used to characterize the genetic basis for the resistance and to determine the location of resistance genes. Treatments had little effect on resistance of the salmonella challenge organism. The greatest resistance to apramycin occurred in E. coli from pigs treated with apramycin and whose sows had earlier exposure to oxytetracycline. Resistance to oxytetracycline was consistently high throughout the study in isolates from all pigs and sows; however, greater resistance was noted in pigs nursing sows that had previous exposure to that drug. The aac(3)-IV gene, responsible for apramycin resistance, was found in approximately 90% of apramycin-resistant isolates and its location was determined to be on plasmids. Several resistant E. coli bio-types were found to contain the resistance gene. These results indicate that resistance to apramycin and oxytetracycline in E. coli of pigs is affected by previous use of oxytetracycline in sows.     

Antimicrobial resistance of Salmonella enterica typhimurium DT104 isolates and investigation of strains with transferable apramycin resistance.

An examination of salmonella isolates collected by the Scottish Agricultural College Veterinary Services Division from April 1994 to May 1995 was conducted to determine the extent to which Salmonella enterica serotype Typhimurium phage type 104 (DT104) occurred and to investigate the antimicrobial resistance patterns of isolates. Typhimurium DT104 was the predominant salmonella and was isolated from nine species of animal. All isolates of this phage type possessed resistance to at least one antimicrobial and 98% of the isolates were resistant to multiple antimicrobials with R-type ACTSp the predominant resistance pattern. Various other resistance patterns were identified and transferable resistance to the veterinary aminoglycoside antimicrobial apramycin was demonstrated in three strains. A retrospective study for gentamicin resistance in isolates from the Scottish Salmonella Reference Laboratory collection revealed a human isolate of Typhimurium DT104 resistant to gentamicin but sensitive to apramycin and a bovine isolate with apramycin and gentamicin resistance.     

Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture

A total of 273 Escherichia coli isolates from raw and treated municipal wastewaters were investigated to evaluate the frequency and persistence of antibiotic resistance and to detect the occurrence of conjugative R plasmids and integrons. The highest resistance rates were against ampicillin (22.71%), tetracycline (19.41%), sulfamethoxazole (16.84%) and streptomycin (14.28%). Multiple antibiotic resistance was present in 24.17% of the isolates. Several multiple antibiotic-resistant isolates proved to be able to transfer en bloc their resistance patterns by conjugative R plasmids with different molecular sizes and restriction profiles. Class 1 integrons of 1 or 1.5 kbp were found in 5 out of 24 representative multiresistant E. coli isolates. Although wastewater treatments proved to be effective in eliminating Salmonella spp. and in reaching WHO microbiological standards for safe use of wastewater in agriculture, they were ineffective in reducing significantly the frequency of plasmid-mediated multiple antibiotic resistance in surviving E. coli. Since multiple antibiotic-resistant bacteria carrying integrons and conjugative R plasmids can constitute a reservoir of antibiotic-resistance genes in wastewater reclaimed for irrigation, risks for public health should be considered. Bacterial strains carrying R plasmids and integrons could contaminate crops irrigated with reclaimed wastewater and transfer their resistances to the consumers' intestinal bacteria.     

Multidrug resistance and plasmid patterns of Escherichia coli O157 and other E. coli Isolated from diarrhoeal stools and surface waters from some selected sources in Zaria, Nigeria

We have assessed the prevalence of Escherichia coli O157 in diarrhoeal patients and surface waters from some selected sources in Zaria (Nigeria), evaluating the antibiotic susceptibility and plasmid profiles of 184 E. coli isolates, obtained from 228 water samples and 112 diarrhoeal stool specimens (collected from children aged <15 years), using standard methods. The detection rate of E. coli O157 in surface waters was 2.2% and its prevalence in children with diarrhoea was 5.4%. The most active antibiotics were gentamicin, chloramphenicol and fluoroquinolones. Seventy-nine (42.9%) of 184 E. coli isolates were resistant to four or more antibiotics. Multidrug resistance (MDR) was higher amongst aquatic isolates than the clinical isolates. Out of 35 MDR isolates (20 of which were O157 strains), 22 (62.9%) harboured plasmids all of which were no less than 2.1 kb in size. Amongst the 20 E. coli O157 strains, only seven (35.0%) contained multiple plasmids. An aquatic O157 isolate containing two plasmids was resistant to seven drugs, including ampicillin, cefuroxime, ciprofloxacin, cotrimoxazole, nalidixic acid, nitrofurantoin and tetracycline. Loss of plasmid correlated with loss of resistance to antibiotics in cured (mutant) strains selected in tetracycline (50 μg/mL)-nutrient agar plates. Our findings revealed that plasmids were prevalent in both the aquatic and clinical isolates, and suggest that the observed MDR is plasmid-mediated. The occurrence of plasmid-mediated multidrug resistant E. coli O157 in surface waters used as sources for drinking, recreation and fresh produce irrigation heightens public health concern.     

Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin in Escherichia coli and Salmonella typhimurium isolated from human and animal sources in Belgium.

Escherichia coli and salmonella strains with plasmids conferring resistance to gentamicin and apramycin have been isolated with increasing frequency both from cattle and hospital patients in Belgium. The apramycin-gentamicin resistance plasmids were characterized in recipient strains by their profiles and molecular weights using agarose gel electrophoresis, by their antimicrobial resistance patterns and by replicon typing using a series of DNA probes specific for the genes controlling their systems of replication. Overall, most of the plasmids differed in their DNA electrophoretic patterns. Seventeen different antimicrobial resistance profiles were observed, and there were six different types of replicons. However, two replication genes predominated and had a preferential distribution in different bacterial species. The rep FIC.a plus rep Q multireplicon was found mainly in plasmids recovered from gentamicin- and apramycin-resistant E. coli while replicon of the type rep FIC.b largely prevailed in S. typhimurium. Identical replication genes were found in most animal and human strains, hence suggesting a high homology between apramycin-gentamicin plasmids in these communities. Finally, our results indicate that the rapid spread of apramycin-gentamicin-resistance in several species of Enterobacteriaceae isolated from animals and from humans in Belgium is not due to a single plasmid, but rather that the gene encoding AAC(3)-IV is carried by various replicons.     

Occurrence of multiple antibiotic resistance and R-plasmids in gram-negative bacteria isolated from faecally contaminated fresh-water streams in Hong Kong

The bacterial populations of six freshwater streams in populated areas of the Hong Kong New Territories were studied. There is considerable faecal contamination of these streams, with coliform counts as high as 10(5) c.f.u./ml and the contaminating organisms show a high prevalence of antibiotic resistance and multiple resistance. With direct plating of water samples onto antibiotic-containing media, an average of 49% of the gram-negative bacteria were ampicillin-resistant, 3% chloramphenicol-resistant and 1% gentamicin-resistant. At individual sites resistance to these drugs was as high as 98%, 8% and 3% respectively. More than 70% of strains were resistant to two or more antibiotics, 29% to five or more and 2% to eight or more. A total of 98 patterns of antibiotic resistance were detected with no one pattern predominating. Twenty-eight gram-negative bacterial species were identified as stream contaminants. Escherichia coli was the commonest bacterial species isolated and other frequent isolates were Enterobacter sp., Klebsiella sp. and Citrobacter sp., but no enteric pathogens were detected. The greatest prevalence of resistance and multiple resistance was associated with the heaviest contamination by E. coli. Analysis of selected stream isolates revealed multiple plasmid bands arranged in many different patterns, but multiple antibiotic resistances were shown to be commonly mediated by single transferable plasmids. Faecally-contaminated freshwater streams in Hong Kong may be reservoirs of antibiotic resistance plasmids for clinically-important bacteria.     

Plasmid-encoded trimethoprim resistance in salmonellas isolated in Britain between 1970 and 1981.

Trimethoprim resistance was plasmid-encoded in all trimethoprim-resistant Salmonella typhimurium and in the majority of trimethoprim-resistant salmonellas of other serotypes isolated since 1970 from humans and food animals in Britain. In S. typhimurium, non-autotransferring plasmids of compatibility group 3 and autotransferring plasmids of group H2 predominated. The predominance of these plasmid types has resulted from the spread of clones of trimethoprim-resistant strains of phage types 18, 170 and 204c. In other salmonellas, a variety of plasmid compatibility groups have been identified. Almost all plasmids which conferred resistance to trimethoprim also coded for sulphonamide resistance.     

Plasmid-encoded trimethoprim resistance in salmonellas isolated in Britain between 1970 and 1981

Trimethoprim resistance was plasmid-encoded in all trimethoprim-resistant Salmonella typhimurium and in the majority of trimethoprim-resistant salmonellas of other serotypes isolated since 1970 from humans and food animals in Britain. In S. typhimurium, non-autotransferring plasmids of compatibility group 3 and autotransferring plasmids of group H2 predominated. The predominance of these plasmid types has resulted from the spread of clones of trimethoprim-resistant strains of phage types 18, 170 and 204c. In other salmonellas, a variety of plasmid compatibility groups have been identified. Almost all plasmids which conferred resistance to trimethoprim also coded for sulphonamide resistance.     

Proteus bacteraemia in a general hospital 1972-1982

During 1972-1982, 318 isolates of Proteus spp. were recovered from blood cultures, accounting for 14.4% of the aerobic, Gram-negative isolates from blood in a large general hospital. Bacteraemia was most common among male patients on the internal medicine service. A high level of resistance to antibiotic agents was found. Since 1976, 49 proteus isolates were sensitive to either gentamicin or tobramycin, but not to both.     

Control of infection with multiple antibiotic resistant bacteria in a hospital renal unit: the value of plasmid characterization.

An outbreak of infections due to multiple antibiotic-resistant bacteria took place over a period of approximately 18 months in a renal unit. Strains of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter spp. and Pseudomonas spp. were involved, and a variety of antibiotic resistances was encountered. Closely related plasmids encoding resistance to aztreonam, ceftazidime and piperacillin, possibly derived from an archetypal plasmid of 105 kb were found in the majority of isolates examined. After limiting the use of aztreonam the incidence of new patient isolates of multiple-resistant organisms was greatly reduced. This study demonstrated how molecular studies can contribute to the control of an outbreak situation in a hospital unit by providing an impetus to reduce the use of specific antibiotics.     

Prevalence of antimicrobial resistance in Enterococcus isolates in Australia, 2005: report from the Australian Group on Antimicrobial Resistance

Antibiotic resistance in Enterococcus species causing clinical disease was examined in a point-prevalence study in 2005. Twenty-two sites around Australia collected up to 100 consecutive isolates and tested them for susceptibility to ampicillin, vancomycin, high-level gentamicin and/or high-level streptomycin using standardised methods. Results were compared to similar surveys conducted in 1995, 1999 and 2003. In the 2005 survey, Enterococcus faecalis (1,987 strains) and E. faecium (180 strains) made up 98.6% of the 2,197 isolates tested. Ampicillin resistance was common (77%) in E. faecium, but rare still in E. faecalis (0.2%). Resistance to vancomycin was 7.2% in E. faecium and 0.2% in E. faecalis; the vanB gene was detected in all vancomycin-resistant isolates. High-level resistance to gentamicin was 35.8% in E. faecalis and 52.2% in E. faecium; the figures for high-level streptomycin resistance were 10.3% and 60.2% respectively. Compared to previous Australian Group on Antimicrobial Resistance surveys in 1995, 1999 and 2003, the proportions of vancomycin resistance and high-level gentamicin resistance in enterococci are increasing. It is important to have an understanding of the occurrence of vancomycin resistant enterococci and high level aminoglycoside resistance in Australia to guide infection control practices, antibiotic prescribing policies and drug regulatory decisions.     

Rapid emergence of ciprofloxacin-resistant enterobacteriaceae containing multiple gentamicin resistance-associated integrons in a Dutch hospital.

In a hematology unit in the Netherlands, the incidence of ciprofloxacin-resistant Enterobacter cloacae and Escherichia coli increased from from 1996 to 1999. Clonal spread of single genotypes of both ciprofloxacin-resistant E. coli and Enterobacter cloacae from patient to patient was documented by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. In addition, genetically heterogeneous strains were isolated regularly. Integrons associated with gentamicin resistance were detected in Enterobacter cloacae and E. coli strains. Integron-containing E. coli were detected in all hematology wards. In contrast, in Enterobacter cloacae strains two integron types were encountered only in the isolates from one ward. Although in all patients identical antibiotic regimens were used for selective decontamination, we documented clear differences with respect to the nosocomial emergence of ciprofloxacin-resistant bacterial strains and gentamicin resistance-associated integrons.