Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Coagulase-negative staphylococci in municipal waste

The total of 259 coagulase-negative staphylococci isolates gained from aerosols created during dumping and utilisation of municipal waste were investigated. Species and subspecies identification allowed to determine predominance of novobiocin-resistant species which primarily colonise rodents or other animals. However, about 30% of isolates were species originating from humans. Metabolic properties of the studied group of isolates were examined, as well as their sensitivity to a set of 8 commonly used antibiotics: amoxicillin, erythromycin, tetracycline, clindamycin, chloramphenicol, gentamicin, trimethoprim/sulfamethoxazole and ciprofloxacin. Many isolates resistant to erythromycin (19%), tetracycline (13%) and a considerably smaller group resistant to clindamycin (6.2%), chloramphenicol (2%) and trimethoprim/sulfamethoxazole (1%) were found. It appeared that processes of occurring at the time of dumping and utilisation of waste affect the prevalence of only some features. The examined metabolic features were found to be relatively stable and no changes were observed, which would indicate the tendency of adaptation to existence in the inanimate matter environment. Only isolates with active nitrate reductase were more frequently detected. The increase in frequency of occurrence of isolates resistant to tetracycline and chloramphenicol and simultaneous elimination of isolates resistant to other antibiotics were observed.     

Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination?

Occurrences of antibiotic-resistant Escherichia coli in two springs of a karstic system (NW France) providing drinking water were determined to study the role of aquifers in the dissemination of the resistance genes. Water samples were collected during wet and dry periods and after a heavy rainfall event to investigate E. coli density, antibiotic resistance patterns, and occurrences of class 1, 2, and 3 integrons. By observing patterns of the resistant isolates (i.e. number and type of resistances) and their occurrences, we were able to define two resistant subpopulations, introduced in the aquifer via surface water: (1) R1-2, characterized by one or two resistance(s), essentially to chloramphenicol and/or tetracycline (96.5%), was always found during the heavy rainfall event; (2) R3-10, characterized by three or more resistances, mostly resistant to tetracycline (94.1%) and beta-lactams (86%), was found transiently. Class 1 and 2 integrons were detected, mostly in the R3-10 subpopulation for class 1 integrons. The characteristics of these two subpopulations strongly suggest that the contamination originates from pasture runoff for the R1-2 subpopulation and from wastewater treatment plant effluents for the R3-10 subpopulation. These two subpopulations of E. coli could be used as biological indicators to determine the origin of groundwater contamination.     

High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from Black-headed Gulls in the Czech Republic

AIMS: To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black-headed Gulls in three nesting colonies. METHODS AND RESULTS: A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29.9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19.1%) isolates. Resistance to ampicillin was detected in 30 (11.7%), cephalothin in 11 (4.3%), streptomycin in 24 (9.3%), sulphonamides in 20 (7.8%) and chloramphenicol in 5 (1.9%) isolates. Nine isolates carrying integrons were detected. CONCLUSIONS: The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. SIGNIFICANCE AND IMPACT OF THE STUDY: Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment.     

Temporal dynamics and impact of manure storage on antibiotic resistance patterns and population structure of Escherichia coli isolates from a commercial swine farm

Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.     

Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla _TEM-1 and bla _CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline‐amended and ciprofloxacin‐amended growth media

Aims: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. Methods and Results: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline‐ (n = 164) and ciprofloxacin‐amended (n = 65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin‐amended growth media (62%) compared to the bacteria isolated on tetracycline‐amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline‐resistant bacteria and almost half of the ciprofloxacin‐resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline‐resistant bacteria was capable of lateral gene transfer. Conclusions: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline‐resistant and ciprofloxacin‐resistant bacteria in municipal wastewater. Significance and Impact of the Study: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.     

Characterization of multiple-antimicrobial-resistant salmonella serovars isolated from retail meats

A total of 133 Salmonella isolates recovered from retail meats purchased in the United States and the People's Republic of China were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and horizontal transfer of characterized antimicrobial resistance determinants via conjugation. Seventy-three (82%) of these Salmonella isolates were resistant to at least one antimicrobial agent. Resistance to the following antibiotics was common among the United States isolates: tetracycline (68% of the isolates were resistant), streptomycin (61%), sulfamethoxazole (42%), and ampicillin (29%). Eight Salmonella isolates (6%) were resistant to ceftriaxone. Fourteen isolates (11%) from the People's Republic of China were resistant to nalidixic acid and displayed decreased susceptibility to ciprofloxacin. A total of 19 different antimicrobial resistance genes were identified in 30 multidrug-resistant Salmonella isolates. The bla(CMY-2) gene, encoding a class A AmpC beta-lactamase, was detected in all 10 Salmonella isolates resistant to extended-spectrum beta-lactams. Resistance to ampicillin was most often associated with a TEM-1 family beta-lactamase gene. Six aminoglycoside resistance genes, aadA1, aadA2, aacC2, Kn, aph(3)-IIa, and aac(3)-IVa, were commonly present in the Salmonella isolates. Sixteen (54%) of 30 Salmonella isolates tested had integrons ranging in size from 0.75 to 2.7 kb. Conjugation studies demonstrated that there was plasmid-mediated transfer of genes encoding CMY-2 and TEM-1-like beta-lactamases. These data indicate that Salmonella isolates recovered from retail raw meats are commonly resistant to multiple antimicrobials, including those used for treating salmonellosis, such as ceftriaxone. Genes conferring antimicrobial resistance in Salmonella are often carried on integrons and plasmids and could be transmitted through conjugation. These mobile DNA elements have likely played an important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.     

Antimicrobial resistance patterns and characterization of integrons of <Emphasis Type="Italic">Shigella sonnei</Emphasis> isolates in Seoul, 1999–2008

A total of 66 Shigella sonnei isolates from 1999 to 2008 in Seoul was analyzed for their antimicrobial resistance, carriage of integron, and the patterns of Pulsed-field gel electrophoresis (PFGE). A high level of antimicrobial resistance to streptomycin (100%), trimethoprim/sulfamethoxazole (95%), tetracycline (94%), nalidixic acid (65%), and ampicillin (41%) was observed among S. sonnei isolates. Fourteen profiles of antimicrobial resistance were identified with the most common resistance profile being nalidixic acid, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole (35%). PCR and DNA sequencing analysis revealed the presence of class 2 integron in all isolates, and class 1 and 2 integrons in 7 isolates. The class 2 integron carried two types of gene cassettes. One cassette array was dfrI, sat2, and aadA1 (91%), and the other was dfr1 and sat1 (8%). dfrA12 and aadA2 gene cassette was found in one isolate containing class 1 integron. PFGE was carried out to examine the genetic relatedness among isolates. All isolates except for one showed similar PFGE patterns (similarity of 80.1%). These results suggest that the S. sonnei isolated during 1999–2008 in Seoul have similar lineages that have not undergone evolutionary changes with time.     

Occurrence and antibiotic sensitivity of Enterobacteriaceae isolated from a group of Jordanian patients with community acquired urinary tract infections

The type and antibiotic sensitivity of urinary tract pathogens may differ in various communities. Of 207 isolates recovered from midstream urine specimens collected from a group of patients with community acquired urinary tract infections (UTI), 86% were species of Enterobacteriaceae. The most frequently recovered pathogens were Escherichia coli (82%), Klebsiella spp. (7.3%), Proteus spp. (6.2%), Enterobacter spp. (3.4%) and Citrobacter spp. (1.1%). High rates of resistance were found against ampicillin (95%), tetracycline (86%), carbenicillin (84%), trimethoprim/sulphamethoxazole (48%), and amoxycillin/clavulanic acid (45%). For the antibiotics tobramycin, aztreonam, ceftriaxone and gentamicin 7% of the isolates were resistant, while resistance varied from 9-18% for amikacin, ciprofloxacin, norfloxacin, nalidixic acid and cefuroxime. The incidence of UTI caused by Enterobacteriaceae was three times higher in females than in males, particularly in young and middle age groups (< or = 19 and 20-39 years).     

Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Novel Method for Rapid Assessment of Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.     

Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment.

A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had "empty" integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTC-resistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.     

Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modified chromogenic agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% +/- 4% overall), sulfamethoxazole, tetracycline (21% +/- 3% overall), and ciprofloxacin (6% +/- 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.     

Detection of Salmonella spp. in retail raw food samples from Vietnam and characterization of their antibiotic resistance

A study was conducted to examine the levels of Salmonella spp. contamination in raw food samples, including chicken, beef, pork, and shellfish, from Vietnam and to determine their antibiotic resistance characteristics. A total of 180 samples were collected and examined for the presence of Salmonella spp., yielding 91 Salmonella isolates. Sixty-one percent of meat and 18% of shellfish samples were contaminated with Salmonella spp. Susceptibility of all isolates to a variety of antimicrobial agents was tested, and resistance to tetracycline, ampicillin/amoxicillin, nalidixic acid, sulfafurazole, and streptomycin was found in 40.7%, 22.0%, 18.7%, 16.5%, and 14.3% of the isolates, respectively. Resistance to enrofloxacin, trimethoprim, chloramphenicol, kanamycin, and gentamicin was also detected (8.8 to 2.2%). About half (50.5%) of the isolates were resistant to at least one antibiotic, and multiresistant Salmonella isolates, resistant to at least three different classes of antibiotics, were isolated from all food types. One isolate from chicken (serovar Albany) contained a variant of the Salmonella genomic island 1 antibiotic resistance gene cluster. The results show that antibiotic resistance in Salmonella spp. in raw food samples from Vietnam is significant.     

Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil

The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.     

A multiresistant clone of Shiga toxin-producing Escherichia coli O118:[H16] is spread in cattle and humans over different European countries

Multiresistant Shiga toxin-producing Escherichia coli (STEC) O118:H16 and O118 nonmotile strains (designated O118:[H16]) were detected by examination of 171 STEC isolates for their antimicrobial sensitivity. Of 48 STEC O118:[H16] strains, 98% were resistant to sulfonamide, 96% were resistant to streptomycin, 79% were resistant to kanamycin, 75% were resistant to tetracycline, 67% were resistant to ampicillin, 60% were resistant to chloramphenicol, 48% were resistant to trimethoprim, and 10% each were resistant to gentamicin and nalidixic acid. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were associated with the mutation gyrA(LEU-83). The STEC O118:[H16] strains were found to belong to a single genetic clone as investigated by multilocus enzyme electrophoresis and by multilocus sequence analysis of E. coli housekeeping genes. The STEC O118:[H16] strains originated from humans and cattle and were isolated in seven different countries of Europe between 1986 and 1999. Strains showing multiresistance to up to eight different antimicrobials predominated among the more recent STEC O118:[H16] strains. The genes in parentheses were associated with resistance to kanamycin (aphA1-Ia), chloramphenicol (catA1), tetracycline [tet(A)], and ampicillin (bla(TEM-1)). Class 1 integrons containing sulI (sulfonamide resistance), aadA1a (streptomycin resistance), or dfrA1 (trimethoprim resistance)-aadA1a gene cassettes were detected in 28 strains. The bla(TEM-1b) gene was present in 18 of 21 strains that were examined by nucleotide sequencing. Class 1 integrons and bla(TEM) genes were localized on plasmids and/or on the chromosome in different STEC O118:[H16] strains. Hybridization of XbaI-digested chromosomal DNA separated by pulsed-field gel electrophoresis revealed that bla(TEM) genes were integrated at different positions in the chromosome of STEC O118:[H16] strains that could have occurred by Tn2 insertion. Our data suggest that strains belonging to the STEC O118:[H16] clonal group have a characteristic propensity for acquisition and maintenance of resistance determinants, thus contrasting to STEC belonging to other serotypes.     

Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France)

Over 6 years, Escherichia coli were isolated from water samples from seven Seine estuary stations, characterized by a densely populated watershed (654 isolates). Resistances of these E. coli to 16 antibiotics were determined and compared with the same resistances in E. coli isolated from a small stream (120 isolates) and from the treated effluent of the largest estuary wastewater treatment plant (123 isolates). Between 30.2% and 56.6% of the estuary isolates were resistant, whatever the station or time of sampling; of these, 60.5–80% were resistant to at least two and up to 12 antibiotics. In the three contrasting sites, resistances to tetracycline, amoxicillin and ticarcillin were the commonest. DNA was extracted from 279 estuary isolates (January 2006) and class 1, 2 and 3 integrons were detected by multiplex real-time PCR and confirmed by classic PCR. IntI1 and intI2 genes were found in 11% of isolates. No intI3 gene was detected. The variable regions of the class 1 and 2 integrons sequenced contained predominantly gene cassettes aadA and dfr . However, for slightly over half of the E. coli isolates exhibiting the class 1 integron, the variable region could not be amplified, because part of the 3' conserved sequence was missing.     

Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes

Aims: To investigate the distribution of the genes that encode enterotoxins and the colonization factors (CF) types as well as the antibiotic susceptibility profile of enterotoxigenic Escherichia coli (ETEC) isolated from children from the Brazilian Northeast. Methods and Results: We conducted a 3·5‐year prospective study that involved 250 children with and 150 without diarrhoea, aged 1–60 months, from low‐income families in Teresina/Brazilian Northeast. All samples were assayed for E. coli, enterotoxin and CF genes and antimicrobial susceptibility by microbiological methods and PCR. ETEC strains were isolated from 9·2% children with and 4·0% without diarrhoea. Infection was more common in children aged 6–24 months in rainy months. elt⁺/CFA/IV⁺ and elt⁺/CS14⁺ were the most frequent genotypes. Susceptibility to nalidixic acid, ciprofloxacin and gentamicin and resistance to ampicillin, cephalothin and sulfamethoxazole–trimethoprim were common. Conclusions: elt ⁺ isolates and ETEC strains harbouring genes encoding CFA/IV and CS/14 were the most common ETEC found in Brazilian Northeast. Significance and Impact of the Study: Our data, the first generated for north‐eastern Brazilian children, may be important for the development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveller’s diarrhoea in the area studied.