Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates

The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA microarray analysis capable of detecting commonly found antimicrobial resistance genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1 integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher in salinomycin-treated chickens than in the control or other treatment groups (P < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production.     

Molecular characterization of antibiotic resistant Escherichia coli strains isolated from tap and spring waters in a coastal region in Turkey

A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.     

Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.     

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.     

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.     

Role of calf-adapted Escherichia coli in maintenance of antimicrobial drug resistance in dairy calves

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 microg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 microg/ml), sulfadiazine (>512 microg/ml), and tetracycline (>4 microg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.     

The Incidence of Antibiotic Resistance among Coliform Bacteria Isolated from Food

Three-hundred-and-seventy-eight strains of coliform bacteria were isolated from specimens of commonly sold milk and food products. Klebsiella and Enterobacter spp. were predominating. Resistance to sulphonamides, streptomycin, and chloramphenicol occurred in only 5, 1, and 2 strains, respectively. No tetracycline-resistant strains were found. Two-hundred-and-two strains (54 %) were resistant to ampicillin. In genetic crosses with a sensitive strain of E. coli Κ 12 W 3132 transmissible R factors could not be demonstrated in any of the resistant coliform strains. It is concluded that food is not a significant source of antibiotic resistant enteric bacteria. It may, however, be suggested that food is a source of potentially pathogenic gram-negative bacteria which points out the importance of strict hygienic surveillance of food production.     

Mathematical model of plasmid-mediated resistance to ceftiofur in commensal enteric Escherichia coli of cattle

Antimicrobial use in food animals may contribute to antimicrobial resistance in bacteria of animals and humans. Commensal bacteria of animal intestine may serve as a reservoir of resistance-genes. To understand the dynamics of plasmid-mediated resistance to cephalosporin ceftiofur in enteric commensals of cattle, we developed a deterministic mathematical model of the dynamics of ceftiofur-sensitive and resistant commensal enteric Escherichia coli (E. coli) in the absence of and during parenteral therapy with ceftiofur. The most common treatment scenarios including those using a sustained-release drug formulation were simulated; the model outputs were in agreement with the available experimental data. The model indicated that a low but stable fraction of resistant enteric E. coli could persist in the absence of immediate ceftiofur pressure, being sustained by horizontal and vertical transfers of plasmids carrying resistance-genes, and ingestion of resistant E. coli. During parenteral therapy with ceftiofur, resistant enteric E. coli expanded in absolute number and relative frequency. This expansion was most influenced by parameters of antimicrobial action of ceftiofur against E. coli. After treatment (>5 weeks from start of therapy) the fraction of ceftiofur-resistant cells among enteric E. coli, similar to that in the absence of treatment, was most influenced by the parameters of ecology of enteric E. coli, such as the frequency of transfer of plasmids carrying resistance-genes, the rate of replacement of enteric E. coli by ingested E. coli, and the frequency of ceftiofur resistance in the latter.     

Characterization of multidrug-resistant Escherichia coli isolates from animals presenting at a university veterinary hospital

In this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection of Escherichia coli isolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1, dfrA1-aadA1, dfrA17-aadA5, dfrA12-orfF-aadA2, bla(OXA-30)-aadA1, aacC1-orf1-orf2-aadA1, dfr7). Class 2 integrons (13.5%) contained the dfrA1-sat1-aadA1 gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected included bla(TEM), cat, floR, aadB, aphA1, strA-strB, sul2, and tet(B), respectively. The bla(CTX-M-2) gene, encoding an extended-spectrum β-lactamase (ESβL), and bla(CMY-2), encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensal E. coli isolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, the bla(CTX-M-2) gene has not previously been reported in Ireland.     

Identification of a new ribosomal protection type of tetracycline resistance gene,tet(36), from swine manure pits

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.     

Serovar distribution and antimicrobial susceptibility of swine Salmonella isolates from clinically ill pigs in diagnostic submissions from Indiana in the United States

Aims:  To determine serovar distribution and levels of antimicrobial susceptibility of Salmonella isolated from clinically ill pigs in diagnostic submissions.
Methods and Results:  A total of 197 Salmonella isolates were obtained by the Indiana Animal Disease Diagnostic Laboratory from 2003 to 2005. Minimal inhibitory concentrations (MICs) were determined using the standard microbroth dilution method. The top four serovars identified were Salm. enterica serovar Typhimurium variant Copenhagen, Salm . Derby, Salm . Choleraesuis var. Kunzendorf and Salm . Typhimurium. All isolates were susceptible to the fluoroquinolones tested except that eight isolates were intermediate to difloxacin. The isolates showed a low prevalence of resistance to trimethoprim/sulphadiazine (Sxt), gentamicin (G), ceftiofur (Cf) and cephalothin (Cp) with low MIC₅₀ value of ≤0·5, 0·5, 1 and 4  μ g ml⁻¹, respectively. They showed a high prevalence of resistance to tetracycline (T; 83·8%), and a moderate prevalence to ampicillin (55·8%), spectinomycin (42·6%), ticarcillin (41·6%) and florfenicol (41·1%). There were more isolates of Salm . Typhimurium, including var. Copenhagen and Salm . Agona, that possessed multiple antimicrobial resistance to amoxicillin/clavulanic acid, ampicillin, ceftiofur and cephalothin (AxApCfCp) than the other serovars.
Conclusions:  The swine Salmonella isolates were susceptible to the fluoroquinolones, Sxt, G, Cf and Cp, but resistant to T.
Significance and Impact of the Study:  These findings provided useful information regarding antimicrobial susceptibility and resistance in dealing with clinical salmonellosis in pig herds.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Antibiotic resistance in bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) from west Wales

The prevalence of antibiotic-resistant bacteria in wild animal and bird populations is largely unknown, with little consistency among the few published reports. We therefore examined intestinal bacteria from magpies ( Pica pica ) and rabbits ( Oryctolagus cuniculus ) collected in rural west Wales. Escherichia coli isolates resistant to multiple antibiotics were grown from eight of 20 magpies trapped in spring, 1999 and one of 17 in spring, 2000; the most prevalent resistance trait among these isolates was to tetracycline, but resistances to ampicillin, chloramphenicol, kanamycin, sulphonamide, tetracycline and trimethoprim were also found. Tetracycline-resistant Enterococcus spp. were found in one of 20 magpies in 1999 and three of 17 in 2000. Only one resistant E. coli isolate was detected among gut bacteria from 13 rabbits, and this strain was resistant only to tetracycline. Differences in the prevalence of resistance between bacteria from rabbits and magpies may reflect differences in diet: rabbits graze field edges, whereas magpies are omnivorous and opportunistic. The resistance genes found in E. coli isolates from magpies mostly corresponded to those common among human isolates, but those conferring tetracycline resistance were unique.     

Comparison of the prevalences and antimicrobial resistances of Escherichia coli isolates from different retail meats in the United States, 2002 to 2008

Escherichia coli isolates were recovered from the National Antimicrobial Resistance Monitoring System retail meat program and examined for antimicrobial susceptibility. Retail meat samples (n = 11,921) from four U.S. states collected during 2002 to 2008, consisting of 2,988 chicken breast, 2,942 ground turkey, 2,991 ground beef, and 3,000 pork chop samples, were analyzed. A total of 8,286 E. coli isolates were recovered. The greatest numbers of samples contaminated with the organism were chicken (83.5%) and turkey (82.0%), followed by beef (68.9%) and pork (44.0%). Resistance was most common to tetracycline (50.3%), followed by streptomycin (34.6%), sulfamethoxazole-sulfisoxazole (31.6%), ampicillin (22.5%), gentamicin (18.6%), kanamycin (8.4%), amoxicillin-clavulanic acid (6.4%), and cefoxitin (5.2%). Less than 5% of the isolates had resistance to trimethoprim, ceftriaxone, ceftiofur, nalidixic acid, chloramphenicol, and ciprofloxacin. All isolates were susceptible to amikacin. Compared to beef and pork isolates, the poultry meat isolates had a greater percentage of resistance to all tested drugs, with the exception of chloramphenicol, to which pork isolates had the most resistance. More than half of the turkey isolates (56%) were resistant to multidrugs (≥3 classes) compared to 38.9% of chicken, 17.3% of pork, and 9.3% of beef isolates. The bla(CMY) gene was present in all ceftriaxone- and ceftiofur-resistant isolates. The cmlA, flo, and catI genes were present in 45%, 43%, and 40% of chloramphenicol-resistant isolates, respectively. Most nalidixic acid-resistant isolates (98.5%) had a gyrA mutation in S83 or D87 or both, whereas only 6.7% had a parC mutation in either S80 or E84. The results showed that E. coli was commonly present in the retail meats, and antimicrobial resistance profiles differed according to the animal origin of the isolates.     

Investigation of Antimicrobial Resistance in Escherichia coli and Enterococci Isolated from Tibetan Pigs

Objectives

This study investigated the antimicrobial resistance of Escherichia coli and enterococci isolated from free-ranging Tibetan pigs in Tibet, China, and analyzed the influence of free-ranging husbandry on antimicrobial resistance.

Methods

A total of 232 fecal samples were collected from Tibetan pigs, and the disk diffusion method was used to examine their antimicrobial resistance. Broth microdilution and agar dilution methods were used to determine minimum inhibitory concentrations for antimicrobial agents for which disks were not commercially available.

Results

A total of 129 E. coli isolates and 84 Enterococcus isolates were recovered from the fecal samples. All E. coli isolates were susceptible to amoxicillin/clavulanic acid, and 40.4% were resistant to tetracycline. A small number of isolates were resistant to florfenicol (27.9%), ampicillin (27.9%), sulfamethoxazole/trimethoprim (19.4%), nalidixic acid (19.4%), streptomycin (16.2%) and ceftiofur (10.9%), and very low resistance rates to ciprofloxacin (7.8%), gentamicin (6.9%), and spectinomycin (2.3%) were observed in E. coli. All Enterococcus isolates, including E. faecium, E. faecalis, E. hirae, and E. mundtii, were susceptible to amoxicillin/clavulanic acid and vancomycin, but showed high frequencies of resistance to oxacillin (92.8%), clindamycin (82.1%), tetracycline (64.3%), and erythromycin (48.8%). Resistance rates to florfenicol (17.9%), penicillin (6.0%), ciprofloxacin (3.6%), levofloxacin (1.2%), and ampicillin (1.2%) were low. Only one high-level streptomycin resistant E. faecium isolate and one high-level gentamicin resistant E. faecium isolate were observed. Approximately 20% and 70% of E. coli and Enterococcus isolates, respectively, were defined as multidrug-resistant.

Conclusions

In this study, E. coli and Enterococcus isolated from free-ranging Tibetan pigs showed relatively lower resistance rates than those in other areas of China, where more intensive farming practices are used. These results also revealed that free-range husbandry and absence of antibiotic use could decrease the occurrence of antimicrobial resistance to some extent.     

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.     

Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.     

The antimicrobial susceptibility of Escherichia coli isolated in eastern area of Romania. A surveillance study

To evaluate the resistance trends for Escherichia coli isolates during 1993-1999 period in Eastern Romania to: ampicillin (A), ampicillin-sulbactam (A/S), ceftazidime (CAZ), cefotaxime (CTA), ceftriaxone (CRO), aztreonam (AZT), ofloxacin (OF), ciprofloxacin (CIP), tetracycline (T) and chloramphenicol (C). We tested 2012 clinical isolates obtained from faeces and urine. MICs were determined by a dilution method in Mueller-Hinton agar (NCCLS guidelines). Resistance rates were analyzed using the NCCLS breakpoints for the fully susceptible category (moderately susceptible strains were classified as resistant). No significant differences were observed in susceptibility of E. coli to ampicillin and ampicillin/sulbactam in the last years. The high percentage of resistant isolates was observed in 1995 for ampicillin (89.7%). Higher incidences of resistance were detected for A, A/S, T; the addition of sulbactam restored A susceptibility only for a small percent. CAZ, CTA, CRO, AZT, OF and CIP resistance among E. coli isolates was progressively increased in the last period.     

Molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Egypt

The aim of this study was to characterize the genetic basis of multidrug resistance in Gram-negative bacteria isolated from bovine mastitis cases in Egypt. Multidrug resistance phenotypes were found in 34 of 112 (30.4%) Gram-negative bacterial isolates, which harbored at least one antimicrobial resistance gene. The most prevalent multidrug-resistant (MDR) species were Enterobacter cloacae (8 isolates, 7.1%), Klebsiella pneumoniae (7 isolates, 6.3%), Klebsiella oxytoca (7 isolates, 6.3%), Escherichia coli (5 isolates, 4.5%), and Citrobacter freundii (3 isolates, 2.7%). The most commonly observed resistance phenotypes were against ampicillin (97.0%), streptomycin (94.1%), tetracycline (91.2%), trimethoprim-sulfamethoxazole (88.2%), nalidixic acid (85.3%), and chloramphenicol (76.5%). Class 1 integrons were detected in 28 (25.0%) isolates. The gene cassettes within class 1 integrons included those encoding resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17, and dfrA25), aminoglycosides (aadA1, aadA2, aadA5, aadA7, aadA12, aadA22, and aac(3)-Id), chloramphenicol (cmlA), erythromycin (ereA2), and rifampicin (arr-3). Class 2 integrons were identified in 6 isolates (5.4%) with three different profiles. Furthermore, the β-lactamase encoding genes, bla(TEM), bla(SHV), bla(CTX-M), and bla(OXA), the plasmid-mediated quinolone resistance genes, qnr and aac(6)-Ib-cr, and the florfenicol resistance gene, floR, were also identified. To the best of our knowledge, the results identified class 2 integrons, qnr and aac(6)-Ib-cr from cases of mastitis for the first time. This is the first report of molecular characterization for antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Africa.