Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter. No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.     

Clinical Evaluation of Co-trimoxazole and Furazolidone in Treatment of Shigellosis in Children

Co-trimoxazole (trimethoprim—sulphamethoxazole) was compared with furazolidone in the treatment of shigellosis in two groups of 33 and 30 patients respectively. Those treated with co-trimoxazole recovered more quickly; none had shigellae in the faeces four days after the start of treatment, whereas in the group given furazolidone eight still had positive stool cultures seven days after treatment.The susceptibility of 104 shigella strains to seven antimicrobial agents was studied by plate dilution technique. All agents but tetracycline and chloramphenicol were found highly effective against most of the strains tested. All shigella isolates were resistant to sulphamethoxazole, and 63% were sensitive to trimethoprim. Potentiation of trimethoprim by sulphamethoxazole was shown in that all strains tested became sensitive to the combination of trimethoprim and sulphamethoxazole in a ratio of 1:20.     

Susceptibility to selected coagulase-negative chemotherapeutic agents of methicillin resistant staphylococci isolated from clinical materials in 1997-1998

The study was aimed at assessment of the sensitivity of methicillin-resistant coagulase-negative staphylococci isolated from clinical material in 1997/1998 to selected chemotherapeutic agents. The investigated material comprised 96 methicillin-resistant coagulase-negative staphylococci from hospital and ambulatory infections isolated during the period from April 1997 to May 1998. Species affiliation was determined by classical identification methods and commercial diagnostic tests for identification of staphylococci. Methicillin resistance was determined by agar disk-diffusion method and screening. Sensitivity to chemotherapeutics was determined by agar disk-diffusion method and agar dilution methods. All the investigated strains were sensitive to nitrofurantoin, furazolidone and vancomycin. To teicoplanin--the second glycopeptide antibiotic--84% strains were sensitive, whereas the percentages of resistant and moderately sensitive strains amounted to 5.2% and 10.4%, respectively. 85% and 82% of coagulase-negative staphylococcal strains were sensitive to fusidic acid and mupirocin. Considerable differences were noted with respect to sensitivity to aminoglycoside group antibiotics. About 35% of strains were sensitive to gentamicin, and 90% sensitive to netilmicin. Ca. 40% of coagulase-negative staphylococci were resistant both to cotrimoxazole and trimethoprim, which, in view of 98% resistance to the second component of cotrimoxazole, may be associated with the activity of only one of the components of the drug--trimethoprim.     

Acute Brucellosis Treated with Trimethoprim and Sulphamethoxazole

Four patients with acute brucellosis are described, none of whom had any connexion with farming or milk industry, the source of infection being different in each case. The diagnosis was made by serological tests, and in three of the four cases was confirmed by positive cultures from bone marrow (one case) and liver biopsy (two cases). Treatment with the combination of trimethoprim/sulphamethoxazole was successful in three out of four cases, and in the fourth case failure may have been due to the development of trimethoprim resistance.     

Susceptibility to selected antibiotics of Yersinia enterocolitica 03 strains, carrying and not carrying plasmid pYV

A total of 199 clinical strains of Yersinia enterocolitica serotype O3, biotype 4 were tested for their susceptibility to antibiotics (158 strains carried the virulence plasmid pYV and 41 strains did not). 114 isolates were tested by standard disk diffusion method for 21 antibiotics. Almost all tested strains were resistant to ampicillin and cefazolin and susceptible to amoxycillin/clavulanate, cefaclor, cefamandole, cefuroxime, cefotaxime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, sulphamethoxazole, co-trimoxazole, trimethoprim and furazolidone. In addition minimal inhibitory concentrations (MICs) of 15 antibiotics were determined by agar dilution method for all 199 strains (158 plasmid positive and 41 strains plasmid negative). Third-generation cephalosporins such as cefotaxime and ceftriaxone and a fluoroquinolone (ciprofloxacin) were the most active antimicrobial agents, tested followed by aztreonam, imipenem, trimethoprim, tetracycline, gentamicin, chloramphenicol, amoxycillin/clavulanate, cefaclor, cefuroxime, amikacin, furazolidone and sulphamethoxazole. The present study demonstrated a high susceptibility of clinical strains of Y. enterocolitica to most of the tested antibiotics. In general, there was no significant difference between susceptibility of virulence plasmid pYV positive and virulence plasmid negative strains to antibacterial agents.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

A simple plate assay for detection of group A streptococcus proteinase

A simple plate assay based upon the digestion of skim milk contained within Columbia agar base medium has been found to be a sensitive and reproducible method for detection of proteinase production by individual surface grown colonies of group A streptococci. Use of this medium enabled demonstration of proteinase activity by 24-h aerobic cultures, 79% of 72 group A streptococcus M prototype strains being proteinase positive. None of 40 ‘viridans’ streptococcus strains were positive on this medium and of 18 representative strains of different Lancefield serogroups, only the group P streptococcus had proteinase activity similar to that of the group A streptococci. This same medium can also be used for the detection of preformed proteinase present in liquid preparations by a well diffusion assay.     

Some Microbiological Aspects of Staphylococcal Mastitis

(1) Mannitol fermentation is a reasonably reliable method for the detection of coagulase positive staphylococci in milk. This reliability can be improved if mannitol fermentation is carried out under anaerobic conditions.(2) Among hemolytic strains of staphylococci isolated from milk, beta hemolytic staphylococci predominate. Bovine and sheep blood agar plates give similar hemolytic patterns, but the hemolysis is more pronounced on sheep blood agar.(3) Gelatin liquefaction cannot be relied upon for the selection of coagulase positive staphylococci in milk.(4) Urease production is a feature of the majority of coagulase positive staphylococci isolated from milk.(5) Tellurite Glycine agar medium is not satisfactory for the selection of coagulase positive staphylococci in milk.     

New medium for rapid screening and enumeration of Clostridium perfringens in foods.

A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM.     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.     

New medium for rapid screening and enumeration of Clostridium perfringens in foods.

A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM.     

High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.     

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for beta-lactamase enzyme. However, 78 (81.25%) of 96 beta-lactamase positive S. aureus strains were beta-lactamase positive ORSA isolates, but none of them had vancomycin resistance.     

Resistogram typing as an epidemiological tool for Escherichia coli isolates of poultry origin

A rapid method for the detection of corynetoxins, tunicamycin-like antibiotics, is described. Test samples were applied to or grown on an agar medium and overlain with Clavibacter tritici which is highly sensitive to the toxins. The method could detect 50 ng of tunicamycin. Corynetoxins in a range of field and laboratory samples were readily detected.     

New Method of Isolating Salmonellae from Milk

The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of <Emphasis Type="Italic">Clostridium difficile</Emphasis> isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Trimethoprim resistance in Finland after five years' use of plain trimethoprim.

A total of 1388 urinary bacterial pathogens were tested for resistance to plain trimethoprim after five years'' use of this drug for prophylaxis against urinary tract infections. Samples were obtained in Turku, Finland, where use of the drug is much greater than in other parts of Finland. Resistance to trimethoprim (greater than 8 mg/l; agar-dilution method) occurred in 20.3% of strains isolated from outpatients and 39.8% of strains isolated from inpatients. Escherichia coli and Micrococcus showed low incidences of resistance (11% and 13% respectively in ouptatients and 23% and 19% respectively in inpatients); Enterobacter, Streptococcus faecalis, and Staphylococcus epidermidis occupied an intermediate position; and Proteus mirabilis and Klebsiella were resistant in 41-76% of cases. Similar incidences of resistance were observed to sulphamethoxazole-trimethoprim, sulphamethoxazole, ampicillin, and nitrofurantoin. These findings together with the rare occurrence of side effects and convenient dosage confirm the usefulness of plain trimethoprim for urinary tract infection.