Antibiotic synergy against biofilm-forming <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Eight antibiotics (aztreonam, ceftazidim, cefoperazon, cefepim, netilmicin, amikacin, ofloxacin and ciprofloxacin) exhibited antimicrobial activity individually and/or in combinations against 20 wild-type biofilm-forming strains of Pseudomonas aeruginosa. The strains were less susceptible in biofilm; in 10 strains antibiotic synergy was observed for the combination of aztreonam and ciprofloxacin. Synergy was also demonstrated in the case of β-lactams and aminoglycosides, β-lactams and fluoroquinolones, aminoglycosides and fluoroquinolones, and for monobactams and β-lactams although the strains were resistant to the individual antibiotics. Synergism or partial synergism was found with one or more antibiotic combinations against 32.4% of isolates.     

Aminoglycoside resistance rates, phenotypes, and mechanisms of Gram-negative bacteria from infected patients in upper Egypt

With the re-emergence of older antibiotics as valuable choices for treatment of serious infections, we studied the aminoglycoside resistance of Gram-negative bacteria isolated from patients with ear, urinary tract, skin, and gastrointestinal tract infections at Minia university hospital in Egypt. Escherichia coli (mainly from urinary tract and gastrointestinal tract infections) was the most prevalent isolate (28.57%), followed by Pseudomonas aeruginosa (25.7%) (mainly from ear discharge and skin infections). Isolates exhibited maximal resistance against streptomycin (83.4%), and minimal resistance against amikacin (17.7%) and intermediate degrees of resistance against neomycin, kanamycin, gentamicin, and tobramycin. Resistance to older aminoglycosides was higher than newer aminoglycosides. The most common aminoglycoside resistance phenotype was that of streptomycin resistance, present as a single phenotype or in combination, followed by kanamycin-neomycin as determined by interpretative reading. The resistant Pseudomonas aeruginosa strains were capable of producing aminoglycoside-modifying enzymes and using efflux as mechanisms of resistance. Using checkerboard titration method, the most frequently-observed outcome in combinations of aminoglycosides with β-lactams or quinolones was synergism. The most effective combination was amikacin with ciprofloxacin (100% Synergism), whereas the least effective combination was gentamicin with amoxicillin (53.3% Synergistic, 26.7% additive, and 20% indifferent FIC indices). Whereas the studied combinations were additive and indifferent against few of the tested strains, antagonism was never observed. The high resistance rates to aminoglycosides exhibited by Gram-negative bacteria in this study could be attributed to the selective pressure of aminoglycoside usage which could be controlled by successful implementation of infection control measures.     

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.     

Association of MexAB-OprM with intrinsic resistance ofPseudomonas aeruginosa to aminoglycosides

MexAB-OprM is known to pump out mostly lipophilic and amphiphilic drugs. But in low-ionic-strength medium, nutrient broth (NB), this pump has been shown to contribute to hydrophilic antibiotic (aminoglycosides) resistance, via active efflux. The association of the MexAB-OprM efflux system to aminoglycosides resistance inPseudomonas aeruginosa were assessed using a drug susceptibility test carried out in NB, in presence and absence of protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) by 23 multidrug resistant strains were selected from 104 clinical isolates ofP. aeruginosa. Active efflux was assessed using EtBr accumulation assays. PCR was used to identify themexAB-oprM and MexAB-OprM-dependent efflux of aminoglycosides and the results were confirmed by continuous fluorescence assay. A multidrug resistant mutant ofmexAB-oprM, derivative of PAO1, was selected by ciprofloxacin and subjected to the same analysis as described above for the clinical isolates. In this study, CCCP reduced the level of MICs in at least 1 dilution. Ethidium bromide accumulation assays confirmed the presence of efflux mechanism in all clinical isolates and PCR demonstrated that 17% of our isolates had themexAB-oprM operon. Results of aminoglycosides accumulation showed, in addition to amphiphilic antibiotics in NB medium, MexAB-OprM extrudes aminoglycosides (hydrophilic) drugs.     

Virulence profiles and other biological characters in water isolated Aeromonas hydrophila

Thirty water isolates of A. hydrophila were tested for potential virulence profiles, antibiotic resistance and Bacteriocin-Like Substances (BLS) production. Cytotoxic activity was present in all strains tested, 87% were hemolytic and 70% adhesive. Lysine decarboxylase reactions (LDC) positivity was correlated with virulence factors: 100% versus cytotoxicity, 84% versus adherence, 76% versus hemolytic activity. The correlation was also present in the LDC-negative strains. Hemolytic and cytotoxic activities were frequently associated: high cytotoxicity, corresponding to high hemolytic activity and vice versa. The in vitro susceptibility of A. hydrophila to 28 antibacterial agents showed that cefotaxime was the most active beta-lactam antibiotic, and Cefuroxime inhibited 90% of the strains. Isolates were resistant to Penicillin G, Ampicillin, Carbenicillin, Amoxicillin, Cephalotin and Cefaclor. Tetracycline, Chloramphenicol, Nitrofurantoine, the quinolones and the aminoglycosides (except Streptomycin) were consistently active. BLS production never emerged against closely-related microorganisms. On the contrary A. hydrophila presented a heteroinhibitory activity against non-taxonomically related genera such as Listeria spp. (L. seeligeri NCTC 11856, L. welshimeri NCTC 11857, L. ivanovii NCTC 11846) and S. aureus ATCC 25923. Although a large number of strains showed virulence determinants together with other biological characters such as antibiotic resistance and BLS production, it was not possible to relate these factors to the observed plasmids.     

In vitro activity of honey,total alkaloids of Sophora alopecuroides and matrine alone and in combination with antibiotics against multidrug-resistant Pseudomonas aeruginosa isolates

Natural products, including honey, total alkaloids of Sophora alopecuroides (TASA) and matrine have been used in combination with antibiotics against various pathogenic bacteria. However, there are limited data on the antibacterial activity of these natural products in combination against multidrug-resistant Pseudomonas aeruginosa strains. The in vitro activity of honey, TASA and matrine alone and in combination with antibiotics against P. aeruginosa isolates was investigated. In this study, four biofilm-producing P. aeruginosa isolates, which were resistant to multiple antibiotics, were used. These natural products were not the most effective single agent against four isolates. The fractional inhibitory concentration index method revealed the synergistic effect of matrine and TASA-honey in combination with ciprofloxacin (Cip) against all tested isolates. When these combinations were used, the resistance of isolates to Cip was decreased significantly (six to eightfold reduction in the minimum inhibitory concentration of Cip. The disk diffusion method showed that all isolates were resistant to β-lactams. Combinations of these antibiotics with TASA and matrine changed slightly the activity of either antibiotic used as a single agent. All isolates produced metallo-β-lactamase enzymes (MBL). Pretreatment isolates with Cip-matrine and Cip-TASA-honey resulted in a statistically downregulated expression of the mexA gene. These natural products can be used against overactivating MexAB-OprM but not MBL-producing P. aeruginosa isolates.     

Comparative antibacterial activity of a novel semisynthetic antibiotic: etimicin sulphate and other aminoglycosides

Etimicin is a novel fourth generation semisynthetic aminoglycoside. It has good antimicrobial activity against both gram-positive and gram-negative bacterial infections and also against aminoglycoside resistant strains. In the present study, in vitro antibacterial activity of etimicin was determined by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill curve tests against type strains and 407 clinical isolates (obtained in a surviellance study), in comparison to other aminoglycosides. Test results revealed that etimicin has potential antimicrobial activity and MIC, MBC values for etimicin were low compared to other aminoglycosides. In MBC test etimicin has exhibited potential bactericidal effect ranging from 0.25 to 2?mg/L. The time kill-curve study further demonstrated the rapid, concentration dependent killing and comparative study showed etimicin to exhibit long and effective bactericidal activity over amikacin. The interesting fact is that most of the tested aminoglycoside resistant clinical isolates were susceptible to etimicin. In view of its potent in vitro antibacterial activity and efficacy profiles, it can be concluded that etimicin can be a potent injectable agent for the treatment of severe bacterial infections.     

Antimicrobial susceptibility of anaerobic bacteria in Bulgaria

ObjectivesThe antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens in the referent for Bulgaria anaerobic laboratory was studied in a period of 25 years/1983–2007/.MethodsNCCLS – recommended agar dilution methods were used. β-lactamase activity was determined with nitrocefin discs.ResultsThe 29 antimicrobial agents included in the study were divided according to their in vitro activity against the anaerobic isolates into 4 main groups for guiding empirical treatment: 1st group of metronidazole, chloramphenicol, meropenem, imipenem and combinations of β-lactam antibiotics with sulbactam – with high activity and drugs of choice for treatment; 2nd group – clindamycin, cefoxitin, carbenicillin/and azlocillin, piperacillin/ – with a good activity and low percent of resistant strains; 3rd group – of tetracycline and erythromycin with higher percent of resistant strains including the new macrolides as josamycin, clarithromycin, roxithromycin and azithromycin; 4th group – penicillins/ampicillin, amoxicillin, penicillin/and cephalosporins/cefamandole, cefazolin, cefotaxime and cefoperazone/ – not suitable for treatment of infections including Bacteroides fragilis group strains, with a very high percent of resistant strains, probably due to β-lactamase activity in most of the strains.ConclusionA continued updating and a follow-up in the changes of antibiotic susceptibility are necessary in every country as resistance patterns vary not only between geographical regions but also even among medical centers and hospitals which may be connected with differences in antibiotic usage in man and animals.     

Combinations of β-Lactam or Aminoglycoside Antibiotics with Plectasin Are Synergistic against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus

Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87–89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections.     

130例嗜麦芽窄食单胞菌下呼吸道感染的药敏分析

目的探讨嗜麦芽窄食单胞菌下呼吸道感染的耐药情况。方法对自2002年5月～2004年5月分离出的130株嗜麦芽窄食单胞菌的药敏试验结果进行分析。结果嗜麦芽窄食单胞菌对常用抗生素广泛耐药,其中对亚胺培南100％耐药,对1、2、3代头孢大多数耐药．但对复方新诺明、替卡西林-克拉维酸钾和头孢他啶的敏感率分别为71．2％、64．0％和54．4%。结论嗜麦芽窄食单胞菌对常用抗生素广泛耐药．但对复方新诺明、替卡西林-克拉维酸钾和头孢他啶有较高的敏感性．可做为临床治疗嗜麦芽窄食单胞菌下呼吸道感染的有效抗菌药物。     

Antibiotic susceptibility, serum response and surface properties of Klebsiella species

Altogether 130 clinical isolates of five Klebsiella species (K. pneumoniae, K. planticola, K. oxytoca, K. ornithinolytica and K. terrigena) were characterized, for their susceptibility to five antibiotics, for susceptibility to serum bactericidal activity and for their hydrophobic properties. All strains exhibited ampicillin resistance. Ampicillin/sulbactam, gentamicin and ofloxacin showed effectiveness in 63.1, 67.7 and 71.5% of the Klebsiella isolates. K. planticola manifested the highest level of resistance to these antibiotics. The majority of Klebsiella strains (93.9%) were susceptible to cefuroxime. Sixty-four strains (49.2%) were serum resistant and intermediate serum sensitivity was shown by 57 strains (43.8%). A high percentage of serum resistant strains (65%) was found in K. planticola. Moderately hydrophobic properties determined by adherence of bacteria to xylene were demonstrated in 25 strains (19.2%).     

Sensitivity assessment of thyme and lavender essential oils against clinical strains of Escherichia coli for their resistance

Strong antiseptic activity of plant essential oils and extracts has been known for a long time. The antibacterial activity of thyme and lavender essential oils were tested against 30 clinical bacterial strains of Escherichia coli from patients with different clinical conditions. The agar diffusion method was used for microbial growth inhibition at various concentrations of the oils from Thymus vulgaris and Lavandula angustifolia. Susceptibility testing to antibiotics and chemotherapeutics was carried out using disc-diffusion method. The results of experiments showed that the both oils, from T. vulgaris and L. angustifolia were active against all of the clinical strains, but thyme oil demonstrated the highest activity. Thyme and lavender essential oils were active against multi drug resistant clinical strains of Escherichia coli genera. The results of experiments justify a study related to activity other essential oils against different genus of bacteria.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Factors Which Influence Synergism by Neomycin and Oxytetracycline

Six strains of enteropathogenic gram-negative bacteria were tested for susceptibility to neomycin or oxytetracycline alone and combined in fixed ratios. The minimal inhibitory concentration for the combination was less than one-half of that expected if the antibiotic activities were simply additive. Neomycin alone was more effective against bacteria multiplying in the presence of abundant oxygen, whereas oxytetracycline alone was more effective against bacteria multiplying in relatively anaerobic environments; when combined, the antibiotics complemented each other by their opposing optima for activity. Oxygen concentration, pH, and neomycin activity are related, and the depression of acid production by oxytetracycline is believed to be partially responsible for the synergistic activity of this pair of antibiotics.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

Therapeutic possibilities in Pseudomonas infections.

Acute pseudomonas infections require treatment with antibiotics producing a bactericidal effect. The most useful are gentamicin, tobramycin, sisomicin and polymyxin B. In resistant strains, amikacin is indicated in addition. Carbenicillin, ticarcillin, carfenicillin or azocillin should never be given alone but in combination with some of the above preparations. Other drugs, such as chloramphenicol, tetracycline or streptomycin, though effective in vitro, should be avoided. Chemotherapy may be complemented by passive immunization either with hyperimmune specific gama globulin or hyperimmune plasma. A programmatic item of combined treatment is active immunization, especially with toxoid vaccine. Chronic processes are not, perhaps with the exception of urinary infections, suitable for antibiotic therapy. For this reason effective polyvalent vaccines should be developed from appropriate strains. It is now certain that in infections caused by mucous strains (most frequently encountered in cystic fibrosis) the vaccine should be prepared from these strains, since they have distinct functional and antigenic characteristics.     

Occurrence of antimicrobial resistance genes sul and dfrA12 in hospital environmental isolates of Elizabethkingia meningoseptica

The aim of this study was to investigate the prevalence, antimicrobial susceptibility and resistant determinants of Elizabethkingia meningoseptica in a Beijing hospital. Four hundred and eighty-seven samples from medical devices, hospital surfaces and medical staff hands were collected. In total, 26 E. meningoseptica isolates were obtained. The sinks, faucets, and drains accounted for more than half of the total number of isolates recovered. Antimicrobial susceptibility testing revealed that 24 isolates were resistant to one or more antibiotics. All strains were susceptible to piperacillin/tazobactam and vancomycin. Although the trimethoprim/sulfamethoxazole has previously been shown to exhibit good activity against E. meningoseptica, in our study 15 strains were resistant to it. We detected trimethoprim/sulfamethoxazole resistance determinants using PCR; six isolates possessed the sulI gene and four possessed the sulII gene, whilst the dfrA12 gene was detected in only one of them. Pulsed-field gel electrophoresis (PFGE) analysis showed 9 distinct types and one dominant pattern with 12 strains was found. Our data indicate that antimicrobial resistant E. meningoseptica strains exist in the hospital environment and susceptibility testing revealed that vancomycin and piperacillin/tazobactam was the most effective antibiotics. These results have practical significance for treatment of E. meningoseptica infection.     

Antimicrobial activity of mupirocin, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline against vancomycin-resistant enterococci (VRE) from clinical isolates in Korea (1998 and 2005)

It is a hot clinical issue whether newly approved antimicrobial agents such as daptomycin, linezolid, quinupristin/dalfopristin (synercid) and tigecycline are active enough to be used for infections caused by vancomycin resistant bacteria. We performed susceptibility tests for mupirocin, which is in widespread clinical use in Korea, and four new antimicrobials, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline, against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium isolated from Korean patients in 1998 and 2005 to evaluate and compare the in vitro activity of these antimicrobials. Among these agents, quinupristin/dalfopristin, which is rarely used in hospitals in Korea, showed relatively high resistance to several vancomycin-resistant enterococci (VRE) isolated in 2005. Likewise, daptomycin, linezolid and tigecycline have not yet been in clinical use in Korea. However, our results showed that most of the 2005 VRE isolates were already resistant to linezolid and daptomycin (highest minimum inhibitory concentration (MIC) value >100 microg/ml). Compared with the other four antimicrobial agents tested in this study, tigecycline generally showed the greatest activity against VRE. However, four strains of 2005 isolates exhibited resistance against tigecycline (MIC >12.5 microg/ml). Almost all VRE were resistant to mupirocin, whereas all E. faecium isolated in 1998 were inhibited at concentrations between 0.8 to approximately 1.6 microg/ml. In conclusion, resistances to these new antimicrobial agents were exhibited in most of VRE strains even though these new antibiotics have been rarely used in Korean hospitals.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

The role of beta-lactamase and the permeability barrier on the activity of cephalosporins against members of the Bacteroides fragilis group

The role of beta-lactamase and the permeability barrier on the activity of some beta-lactams against 53 strains of the Bacteroides fragilis group was investigated. Minimal inhibitory concentrations of cefamandole, cefoxitin, and cephalothin were determined with or without the addition of clavulanic acid and (or) ethylenediaminetetraacetate using an agar dilution technique. A significant increase of susceptibility with clavulanic acid indicated a role for beta-lactamase, and with ethylenediaminetetraacetate, a role for a permeability barrier. We found that both beta-lactamase and low permeability decreased the activity of the beta-lactams to some extent depending on the bacterial species and the antibiotic. The species-specific exception was B. distasonis which showed only a permeability barrier to all antibiotics tested.