Antimicrobial susceptibility testing of bacteria isolated from animals: considerations concerning the predefinition of breakpoints from the clinical pharmacological viewpoint

In vitro susceptibility tests are performed to receive information for selecting the most suitable antibacterial agent. As result of in vitro susceptibility tests, the minimum inhibitory concentration (MIC) indicates bacteria as resistant or sensitive. To determine MIC, therapeutically relevant breakpoints have to be defined. Microbiological criteria, chemical and physical characteristics as well as pharmacokinetic and toxicological (tolerance) properties of the antimicrobial compounds have to be considered in the selection of the therapeutic agent in addition to clinical experiences. Using some concentration and time dependent antibiotics as examples, it is demonstrated that the above mentioned criteria are not sufficiently considered in currently defined breakpoints.     

Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus pseudintermedius isolated from various animals

This study characterized the antimicrobial susceptibility of 221 Staphylococcus aureus isolated from various species, and 60 canine Staphylococcus pseudintermedius isolated from 1986 through 2000 at the Western College of Veterinary Medicine (WCVM). Resistance of S. aureus was most common to penicillin (31%) and tetracycline (14%); resistance of S. pseudintermedius to penicillin was present in 8% and to tetracycline in 34% of isolates. Resistance to trimethoprim/sulfamethoxazole was only seen among S. pseudintermedius, and there was no resistance to amoxicillin/clavulanate, ampicillin/sulbactam, cephalothin, amikacin, gentamicin, enrofloxacin, chloramphenicol, or rifampin among any isolate. Inducible clindamycin resistance was found in both S. aureus and S. pseudintermedius, highlighting the need for careful interpretation of culture and susceptibility test results. There were significant differences in the minimum inhibitory concentrations of penicillin, ciprofloxacin, enrofloxacin, clindamycin, erythromycin, chloramphenicol, and tetracycline between avian, bovine, equine, and porcine isolates.     

Antimicrobial resistance in Salmonella isolated from animals and their environment in England and Wales from 1988 to 1999

Resistance to 16 antimicrobial agents was monitored in 109,125 Salmonella cultures isolated from animals, their environment and feedstuffs between 1988 and 1999. The sensitivity of the 6512 isolates of Salmonella enterica enterica serotype Dublin to all the antimicrobial agents tested varied from 98.2 per cent in 1997 to 99.7 per cent in 1990 and 1996. In contrast, among 28,053 isolates of Salmonella enterica enterica serotype Typhimurium, there was a marked decrease in their sensitivity to all the antimicrobial agents tested, from 57.4 per cent in 1992 to 7.6 per cent in 1995, owing to the widespread occurrence in farm animals of S Typhimurium isolates of the definitive type DT104, resistant to ampicillin, sulphonamides, streptomycin, chloramphenicol and tetracyclines, although the percentage of sensitive isolates increased to 18.4 per cent in 1999, when the incidence of DT104 had decreased. Some isolates of DT104 also showed an increase in resistance to potentiated sulphonamides (2.4 per cent in 1989 to 19.2 per cent in 1999) and nalidixic acid (0 per cent in 1992, 3.8 per cent in 1995 to a peak of 16.9 per cent in 1998). In 1996, 5.1 per cent of 1086 isolates of S Typhimurium from cattle and 35.9 per cent of 192 isolates of S Typhimurium from poultry showed resistance to nalidixic acid. Of the other 74,528 Salmonella isolates, the percentage of strains sensitive to all the antimicrobials tested decreased slightly from 88.2 per cent in 1988 to 70.6 per cent in 1996 and then increased slightly to 73.7 per cent in 1999. The commonest of these other Salmonella serotypes was Salmonella Enteritidis (20,982), which remained predominantly susceptible (ranging from 81.4 to 97.4 per cent) during the study period. Few isolates were resistant to commonly used veterinary antimicrobials, for example, furazolidone, the use of which was banned in 1990, and the aminoglycoside, apramycin.     

Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods

In the present review, a new mass screening system for selecting probiotic strains from Lactobacillus (L) acidophilus group lactic acid bacteria (LAB) with strong adhesion to the human intestinal tract is described. Characteristics of antimicrobial peptides (bacteriocin), lactose‐hydrolyzing enzymes and immunostimulative oligo DNA motifs in L. gasseri strains are also described. Finally, the use of L. acidophilus LAB, selected by our screening method, that have strong adhesion to the human colonic mucosa in functional yogurt products is described. Adhesiveness to the human intestine is one of the most important characteristics of probiotic LAB. A new screening system that involves a combination of three methods is proposed: rat colonic mucin (RCM)‐micro plate assay, Carnoy's histochemical staining method and carbohydrate probe binding assay. By using an RCM‐coated poly‐vinylidene‐diflouride membrane that mimics the human colonic mucous layer, a new lectin was isolated and its structure was clarified by gene cloning. Furthermore, the structures and functions of a new cyclic bacteriocin (gassericin A), new lactose‐hydrolyzing enzymes, and new immunostimulating oligo DNA motifs from Lactobacillus gasseri (B₁ subgroup) were clarified. A new functional yogurt ‘Fit down’ is proposed, that is fermented by an adhesive strain of L. acidophilus LA67 selected by our screening and contains antihypertensive peptides derived from whey proteins by protease digestion. In the future, superior functional foods containing more effective probiotic LAB are expected to be developed by the use of the proposed mass screening system.     

Staphylococci isolated from animals and food with phenotypically reduced susceptibility to beta-lactamase-resistant beta-lactam antibiotics

The antibiotic resistance pattern of 1921 Staphylococcus strains isolated from animals and food within the last two years were examined using diffusion tests. Among them there were only 35 strains of S. aureus having an inhibition zone diameter of 15 mm or less, and 4 strains of coagulase-negative staphylococci (CNS) having a zone diameter of 18 mm or less to 1-microg oxacillin disk. These 39 strains were examined also by E-test to oxacillin and for the detection of the mecA gene by PCR in order to determine whether they might be real methicillin-resistant staphylococci. Among the 39 strains there were only two that were susceptible to penicillin by disk diffusion method; however, further examination by the penicillinase test showed that they produced beta-lactamase. While 19 (15 S. aureus, 4 CNS) strains were resistant and 7 strains were intermediate to oxacillin in disk diffusion test, the E-test gave 8 resistant and 5 intermediate results. Six out of the 8 oxacillin-resistant strains examined by disk diffusion and E-test harboured the mecA gene. Thus only 6 out of the examined 1921 strains proved to be mecA positive. These methicillin-resistant, mecA-positive strains (5 of the S. aureus strains and 1 of the S. epidermidis) originated from two dairy herds. The results prove that methicillin-resistant S. aureus (MRSA) strains in animals are really rare in Hungary. Eighteen strains were chosen and screened for minimal inhibitory concentration (MIC) of oxacillin with or without clavulanic acid or sulbactam, and three of them produced methicillinase enzyme.     

Antimicrobial drug susceptibility of bacteria isolated from disease processes in cattle, horses, dogs and cats

Results are presented of antimicrobial disc diffusion susceptibility testing on commonly isolated bacterial pathogens made at the Ontario Veterinary College Diagnostic Bacteriology Laboratory in 1981 and 1982. Nearly 2 000 isolates from horses, cattle, dogs and cats were tested. Comparison of resistance in the same bacterial species isolated from different animal species showed significant differences between some of the same antibiotics.     

Antimicrobial susceptibility of bacteria isolated from neonatal foal samples submitted to a New Zealand veterinary pathology laboratory (2004 to 2013)

AIMS: To describe antimicrobial susceptibility, and identify antimicrobial resistance (AMR), in bacteria isolated from New Zealand foals.

METHODS: A database search was performed of submissions to a veterinary pathology laboratory between April 2004 and December 2013 for bacterial culture of samples from foals <3 weeks of age. Culture and susceptibility results were compiled with demographic information. Susceptibility results were as defined for the Kirby-Bauer disk diffusion susceptibility test based on Clinical Laboratory Standards Institute guidelines. Multi-drug resistance (MDR) was defined as non-susceptibility to ≥3 of a panel of antimicrobials (ceftiofur, enrofloxaxin, gentamicin, penicillin, tetracycline, trimethoprim-sulfonamide); penicillin susceptibility was not included for Gram-negative isolates.

RESULTS: Submissions from 102 foals were examined, and 127 bacterial isolates were cultured from 64 (63%) foals. Of the 127 isolates, 32 (25%) were Streptococcus spp., 30 (24%) were Staphylococcus spp., 12 (10%) were Enterococcus spp. and 26 (21%) were Escherichia coli. Of 83 Gram-positive isolates, 57 (69%) were susceptible to penicillin. Over all isolates, 92/126 (73%) were susceptible to gentamicin and 117/126 (93%) to enrofloxacin; 62/82 (76%) of Gram-positive, and 22/42 (52%) of Gram-negative bacteria were susceptible to ceftiofur; 53/81 (65%) of Gram-positive, and 23/44 (52%) of Gram-negative bacteria were susceptible to tetracycline; 59/82 (72%) of Gram-positive, and 23/44 (43%) of Gram-negative bacteria were susceptible to trimethoprim-sulfonamide. Of 126 isolates, 33 (26%) had MDR; >1 isolate with MDR was cultured from 24/64 (38%) foals, and ≥2 isolates with MDR were recovered from 8/64 (13%) foals.

CONCLUSIONS: Multi-drug resistance, including resistance to commonly used antimicrobials, was found in bacterial isolates from foals in New Zealand.

CLINICAL RELEVANCE: The results of this study are of concern from a treatment perspective as they indicate a potential for antimicrobial treatment failure. For future surveillance of AMR and the creation of national guidelines, it is important to record more data on samples submitted for bacterial culture.     

Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment.

The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 microg/ml; ceftiofur was 32 microg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 microg/ml; nalidixic acid, nitrofurantoin, and sulfamethoxazole were 128 microg/ml; amikacin, ampicillin, gentamicin, lincomycin, spectinomycin, streptomycin, tetracycline, and trimethoprim were > or = 256 microg/ml. The therapeutic efficacy of ceftiofur against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental ducks were infected through the infraorbital sinus with 1 ml of 9 x 10(9) CFU of R. anatipestifer. Ceftiofur (0, 0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.     

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.     

Survey of marbofloxacin susceptibility of bacteria isolated from cattle with respiratory disease and mastitis in Europe

A monitoring programme conducted in Europe since 1994 to survey the marbofloxacin susceptibility of bacterial pathogens isolated from cattle has established the susceptibility of bacterial strains isolated before any antibiotic treatment from bovine mastitis and bovine respiratory disease (BRD) cases between 2002 and 2008. Minimum inhibitory concentration (MIC) was determined by a standardised microdilution technique. For respiratory pathogens, Pasteurella multocida and Mannheimia haemolytica isolates (751 and 514 strains, respectively) were highly susceptible to marbofloxacin (MIC≤0.03 μg/ml for 77.39 per cent of the strains) and only 1.75 per cent of M haemolytica strains were resistant (MIC≥4 μg/ml). Histophilus somni isolates (73 strains) were highly susceptible to marbofloxacin (0.008 to 0.06 μg/ml). Mycoplasma bovis MIC (171 strains) ranged from 0.5 to 4 μg/ml. For mastitis pathogens, the majority of Escherichia coli isolates were highly susceptible to marbofloxacin (95.8 per cent of 617 strains). Staphylococcus aureus and coagulase-negative staphylococci (568 and 280 strains) had a homogenous population with MIC centred on 0.25 μg/ml. Streptococcus uberis and Streptococcus dysgalactiae (660 and 217 strains) were moderately susceptible with MIC centred on 1 μg/ml. Marbofloxacin MIC for these various pathogens appeared stable over the seven years of the monitoring programme and was similar to previously published MIC results.     

Molecular analysis of the virulence determinants of enterotoxigenic Escherichia coli isolated from domestic animals: applications for vaccine development

Enterotoxigenic strains of Escherichia coli are an important cause of diarrhoeal disease in young farm animals. Several virulence determinants have been shown to play a major role in the pathogenicity of these strains. The molecular structure of some of these determinants including adhesion fimbriae, heat-labile toxins and heat-stable toxins have been elucidated. This knowledge has made possible the development of novel vaccines effective against enterotoxigenic strains. In this short review, the structure of these virulence factors will be described and the implications for the development of future vaccines will be discussed.     

Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa

The bacterial and fungal flora of the external ear canal of dogs with otitis externa and of healthy dogs were studied. The most frequently isolated microorganism from otitic ears was Staphylococcus intermedius (58.8%), followed by Malassezia pachydermatis (30.9%), Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%). A statistical analysis of our results showed that the prevalence of these microorganisms is significant in dogs with otitis externa. Furthermore, the antimicrobial susceptibility patterns of isolated strains were determined. Majority of all bacterial isolates were most susceptible to gentamicin. Malassezia pachydermatis, the most prevalent yeast in this study, showed an excellent level of susceptibility to all antifungal agents tested.     

奶牛乳房炎病原菌的分离鉴定及药敏试验

针对郑州某一奶牛小区进行调查,从40个乳样中分离到14种共67个菌株,其中无乳链球茵15株,占22．39％,停乳链球菌14株,占20．90％；葡萄球菌14株,占20．90％；大肠杆菌7株,占10．45％。药敏试验结果表明,环丙沙星、头孢唑啉等药物有良好的抑菌效果,而病原菌对青霉素、链霉素已有很大药性。     

Antimicrobial susceptibility of Bordetella bronchiseptica isolates from cats and a comparison of the agar dilution and E-test methods

One hundred and fifty-two predominantly feline isolates of Bordetella bronchiseptica were tested for their susceptibility to seven antimicrobial agents using an agar dilution method. The majority of isolates tested by the agar dilution method were resistant to trimethoprim (MIC₉₀ 500 μg/ml) and ampicillin (MIC₉₀ > 32 μg/ml) but sensitive to tetracycline, doxycycline and enrofloxacin (MIC₉₀ 2 μg/ml for all three agents). The isolates showed a spectrum of susceptibility to sulphadiazine and clavulanate potentiated amoxycillin. The MIC's of twenty-nine of the 152 isolates were then compared for five of the antimicrobial agents using the E-test (AB Biodisk, Sweden), a recently introduced method for measuring the MIC's of antimicrobial agents based on the diffusion of a pre-defined antibiotic gradient from a plastic strip. Comparisons with the E-test demonstrated an overall agreement (±1 log₂ dilution) with the agar dilution method of 79.4% and an agreement within ±2 log₂ dilutions of 96.2%.     

Use of antimicrobial susceptibility testing of bacterial pathogens isolated from the milk of dairy cows with clinical mastitis to predict response to treatment with cephapirin and oxytetracycline

OBJECTIVE: To determine whether results of antimicrobial susceptibility testing of bacterial pathogens isolated from the milk of dairy cows with clinical mastitis were associated with duration of clinical signs or bacteriologic cure rate following treatment with cephapirin and oxytetracycline. DESIGN: Observational study on a convenience sample. ANIMALS: 58 dairy cows with 121 episodes of clinical mastitis. PROCEDURE: Cows that only had abnormal glandular secretions were treated with cephapirin alone. Cows with an inflamed gland and abnormal glandular secretions were treated with oxytetracycline and cephapirin. Cows with systemic signs of illness, an inflamed gland, and abnormal glandular secretions were treated with oxytetracycline and flunixin meglumine and frequent stripping of the affected glands. The Kirby-Bauer method was used for antimicrobial susceptibility testing, and current guidelines were used to categorize causative bacteria as susceptible or resistant to the treatment regimen. RESULTS: Median durations of episodes of clinical mastitis caused by susceptible (n = 97) and resistant (24) bacteria were not significantly different. Bacteriologic cure rates at 14 and 28 days were similar for episodes caused by susceptible and resistant bacteria; however, for 56 episodes of clinical mastitis caused by gram-positive bacteria and treated with cephapirin alone, bacteriologic cure rate at 28 days was significantly higher for susceptible than for resistant bacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that antimicrobial susceptibility testing was of no value in predicting duration of clinical signs or bacteriologic cure rate in dairy cows with mastitis, except for episodes caused by gram-positive organisms treated with intramammary administration of cephapirin alone.     

Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.     

Antimicrobial activity of bovine bactericidal permeability-increasing protein-derived peptides against gram-negative bacteria isolated from the milk of cows with clinical mastitis

OBJECTIVE: To evaluate antimicrobial activity of bovine bactericidal permeability-increasing protein (bBPI)-derived synthetic peptides against mastitis-causing gram-negative bacteria. SAMPLE POPULATION: Bacterial isolates from the milk of cows with clinical mastitis. PROCEDURES: 3 peptides were synthesized with sequences corresponding to amino acids 65 to 99 (bBPI(6,599)) or 142 to 169 (bBPI(142,169)) or the combination of amino acids 90 to 99 and 148 to 161 (bBPI(9,099, 148,161)) of bBPI. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these peptides against bacterial isolates from cows with mastitis were determined by use of a standardized broth microdilution assay. The ability of these peptides to retain their antimicrobial activity in serum and milk was also evaluated. Finally, bacterial lipopolysaccharide (LPS)-neutralizing activity of these peptides was assayed with the Limulus amebocyte lysate test. RESULTS: Of the 3 peptides tested, bBPI(9,099, 148,161) had the widest spectrum of antimicrobial activity, with MIC and MBC values ranging from 16 to 64 Mg/mL against Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp and from 64 to 128 Mg/mL against Pseudomonas aeruginosa. None of the peptides had any growth-inhibitory effect on Serratia marcescens. The antimicrobial activity of bBPI(9,099, 148,161) was inhibited in milk, but preserved in serum. Finally, bBPI(142,169) and bBPI(9,099, 148,161) completely neutralized LPS. CONCLUSIONS AND CLINICAL RELEVANCE: bBPI(9,099, 148,161) is a potent neutralizer of the highly proinflammatory molecule bacterial LPS and has antimicrobial activity against a variety of gram-negative bacteria. The ability of bBPI(9099,148161) to retain antimicrobial activity in serum suggests a potential therapeutic application for this peptide in the management of gram-negative septicemia.     

Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization

Fourteen and 22 each of Salmonella Enteritidis and Salmonella Typhimurium (S. Typhimurium) were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns, phage types and resistance gene patterns. S. Typhimurium isolates were highly resistant to streptomycin, sulfisoxazole and tetracycline, 95, 95 and 86%, respectively. The incidence of multiple antibiotic resistance (resistant to more than two drugs tested) of S. Typhimurium isolates was extremely high (100%) comparing to S. Enteritidis isolates (21%). Two of the five ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline) resistant type S. Typhimurium isolates were phage type definitive type 104 (DT104).For the detection of resistance related genes in S. Enteritidis and S. Typhimurium isolates, particularly ACSSuT type S. Typhimurium, antibiotic resistance genes, cmlA/tetR, bla(PSE-1) and bla(TEM), and genus Salmonella specific gene, sipB/C, were amplified using four pairs of primers in a hot-start multiplex polymerase chain reaction (PCR). Two Korean isolates of S. Typhimurium DT104 showed bla(TEM) amplicons instead of bla(PSE-1) for the ampicillin resistance and they were susceptible to florfenicol. The multiplex PCR used in this study was useful in characterization of multiple drug resistant Salmonella isolates, especially ACSSuT type S. Typhimurium, and identification of beta-lactamase gene distribution among Salmonella isolates.