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.     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

Screening of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal strains isolated from medical students for toxin genes

Three hundred twenty-one students (156 students with no clinical exposure and 165 students with clinical exposure) were screened for nasal colonization by Staphylococcus aureus; 20.9% of students were S. aureus nasal carriers, and 40.3% of S. aureus isolates harbored toxin genes. The most prevalent genes were tst (15.0 %) and sec (13.4 %). Isolates with multiple genes were only found among clinical students (p = 0.045). Six of 11 PFGE clones were positive for toxin genes. Methicillin-resistant (MRSA) isolates were only detected in the clinical students (4.5 %). The exposure of students to the hospital environment neither radically increased S. aureus nasal carriage, nor the frequency of clinically important toxin gene presence, but it could have influenced the positive selection of toxigenic MRSA strains.     

Typing of Staphylococcal Cassette Chromosome <Emphasis Type="Italic">mec</Emphasis> Encoding Methicillin Resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Strains Isolated at the Bone Marrow Transplant Centre of Tunisia

Staphylococcal Cassette Chromosome mec (SCCmec) is a mobile genetic element that carries the gene mecA mediating the methicillin resistance in staphylococci. It is composed of mec and ccr gene complexes. Six SCCmec types have been defined so far. SCCmec typing of 13 methicillin-resistant Staphylococcus aureus (MRSA) out of 72 (18%) non redundant S. aureus strains recovered in 1998–2007 at the Bone Marrow Transplant Centre of Tunis was carried out. The isolates were identified by conventional methods. Antibiotic susceptibility was determined by oxacillin and cefoxitin disks and oxacillin MIC by E-test. Methicillin resistance was detected by mecA PCR. The SCCmec complex types were determined by PCR. The epidemiology of MRSA has been investigated by PFGE. Among 13 mecA positive strains, 12 were resistant to oxacillin (MIC = 3 to >256 μg/μl) and to cefoxitin and one strain was pre-resistant: susceptible to oxacillin (MIC = 0.19 μg/μl) and to cefoxitin. Hospital-acquired MRSA (HA-MRSA) strains had essentially SCCmec type IV (nine strains) or III (two strains) or I (one strain). One strain shown to carry ccrAB1 and ccrAB2 genes in combination with class B mec. Seven of 13 MRSA strains isolated from 2000 to 2006 were classified with major similarity group A harbored SCCmec type IV.     

Antibacterial Effects of Green Tea Polyphenols on Clinical Isolates of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antibacterial effects of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. Characterization of the minimal inhibitory concentration (MIC) of oxacillin for 30 S. aureus strains isolated from patients treated with oxacillin identified 13 strains with an oxacillin MIC ≥ 4 μg/mL as methicillin-resistant Staphylococcus aureus (MRSA) (range: 8 to 512 μg/mL), while 17 strains were methicillin-susceptible Staphylococcus aureus (MSSA) (range: 0.25–0.5 μg/mL). The MICs of TPP ranged from 50 to 180 μg/mL for both the MSSA and the MRSA strains. The MICs of oxacillin for each of the 13 MRSA strains were reduced between 8- and 128-fold when these strains were coincubated with sub-MIC (≤0.5× MIC) levels of TPP, demonstrating that the combination of TPP plus oxacillin was synergistic for all of the clinical MRSA isolates. Two-dimensional polyacrylamide gel electrophoresis identified 14 extracellular proteins of MRSA-13 down-regulated and 3 proteins up-regulated by exposure to TPP. These studies demonstrate that TPP can differentially stimulate the expression of various proteins in these bacteria and synergize the bactericidal activity of oxacillin for MRSA.     

Detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using double duplex real-time PCR and dye Syto 9

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) using real-time polymerase chain reaction (PCR) and dye Syto 9 was developed and evaluated. The assay was based on the two duplex reactions run simultaneously. The detection reaction amplified staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and S. aureus-specific 442-bp DNA (Sa442). The control reaction amplified S. aureus-specific nuclease gene nuc and a marker of methicillin resistance, mecA. The method was evaluated by analyzing 214 clinical S. aureus isolates yielding 98.7 % sensitivity, 100 % specificity, 100 % positive predictive value and 96.6 % negative predictive value for detection of MRSA. The detection limit was determined to be 15–80 genome copies per real-time PCR. It was able to discriminate between MRSA, methicillin resistant coagulase negative staphylococci and methicillin susceptible S. aureus (MSSA) isolates containing only small fragments of the right extremity of the SCCmec (MSSA revertants).     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

SCC<Emphasis Type="Italic">mec</Emphasis> Type IV,PVL-Negative,Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Cystic Fibrosis Patients from Brazil

Twenty seven S. aureus isolates were obtained from cystic fibrosis (CF) patients at a tertiary care hospital in Brazil. Nineteen (70.4%) were methicillin-susceptible S. aureus (MSSA) and eight (29.6%) methicillin-resistant S. aureus (MRSA). Of the MRSA isolates, four had SCCmec type III and four had SCCmec type IV. PVL genes were not detected in any of the MSSA or MRSA isolates. New studies are necessary to evaluate the exact impact of these different MRSA clones in CF patients.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

The catalase gene differentiates between some strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> ssp. <Emphasis Type="Italic">anaerobius</Emphasis>

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99 % identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from “C” in katB to “T” in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.     

Vancomycin-resistance Transferability from VanA Enterococci to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In last decade methicillin-resistant Staphylococcus aureus with high level of vancomycin-resistance (VRSA) have been reported and generally the patients with VRSA infection were also infected with a vancomycin-resistant Enterococcus (VRE). Considering that the high level of vancomycin-resistance in VRSA isolates seems to involve the horizontal transfer of Tn1546 transposon containing vanA gene from coinfecting VRE strains, the authors have studied the “in vitro” conjugative transfer of this resistance from VanA enterococci to S. aureus. Out of 25 matings performed combining five vancomycin-resistant enterococci as donors (three Enterococcus faecalis and two Enterococcus faecium), and five S. aureus as recipients, all clinical isolates, two have been successful using E. faecalis as donor. The transfer of vancomycin-resistance was confirmed by vanA gene amplification in both transconjugants and the resistance was expressed at lower levels (MIC 32 μg/ml) in comparison with the respective VRE donors (MIC > 128 μg/ml). The vancomycin-resistance of trasconjugants was maintained even after subsequent overnight passages on MSA plates containing subinhibitory levels of vancomycin. This study shows that the vanA gene transfer can be achieved through techniques “in vitro” without the use of laboratory animals employed, in the only similar experiment previously carried out by other authors, as substrate for the trasconjugant growth. Moreover, in that previous experiment, contrary to this study, the vancomycin resistant S. aureus trasconjugants were selected on erythromycin agar and not by direct vancomycin agar selection.     

Synergistic effect between dieckol from <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> and β-lactams against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We have been attempting for some time to discover a compound evidencing antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The dieckol isolated from Ecklonia stolonifera has been shown to exhibit antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA. The minimum inhibitory concentrations (MICs) of dieckol were determined in a range of 32 to 64 μg/mL against standard MSSA and MRSA strains. Furthermore, dieckol clearly reversed the high-level ampicillin and penicillin resistance of MRSA. The MICs of ampicillin against two standard strains of MRSA were dramatically reduced from 512 to 0.5 μg/mL in combination with 1/4 MIC of dieckol (16 μg/mL). The fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were measured from 0.066 to 0.266 in combination with 8 or 16 μg/mL of dieckol against all tested MRSA strains, thereby suggesting that dieckol-ampicillin or dieckol-penicillin combinations exert a synergistic effect against MRSA. The results of this study indicate that dieckol, administered in combination with β-lactams, may prove effective in the treatment of MRSA infections. Our finding may also contribute to the development of an alternative phytotherapeutic anti-MRSA agent.     

High-Level Oxacillin and Gentamycin Resistance with Reduced Susceptibility to Vancomycin in <Emphasis Type="Italic">Staphylococcus aureus-</Emphasis>Carrying <Emphasis Type="Italic">mecA</Emphasis> and <Emphasis Type="Italic">femA</Emphasis> Gene Complex

Staphylococcus aureus oxiva 10 and oxiva 14 strains clinically isolated from diabetic patients were resistant to gentamycin and oxacillin The minimal inhibitory concentrations (MICs) of oxacillin and gentamycin were 720 and >2048 μg/mL, respectively, for oxiva 10 and 680 and 400 μg/mL. respectively, for oxiva 14; both strains carry mecA and femA genetic determinants in their genomes. In addition, both are vancomycin-intermediate Staphylococcus aureus (VISA) isolates. The addition of vancomycin led to significant decreases in oxacillin resistance of both oxiva 10 and oxiva 14 strains, whereas the addition of vancomycin to gentamycin plates showed a decrease in gentamycin resistance of non-high-level gentamycin-resistant (non-HLGR) oxiva 14 and indifference in gentamycin resistance in HLGR oxiva 10. Transmission electron microscopy of representative strains unveils a remarkable increase in the thickness of the cell wall, indicating that thickening of the cell wall is a common phenotype associated with vancomycin resistance in VISA isolates. The present study reports that the rate of synergism and synergistic effect in the combination vancomycin–gentamycin vary according to the MICs of gentamycin.     

Efficacy of Targeted 5-day Combined Parenteral and Intramammary Treatment of Clinical Mastitis Caused by Penicillin-Susceptible or Penicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Combined parenteral and intramammary treatment of mastitis caused by Staphylococcus aureus was compared to parenteral treatment only. Cows with clinical mastitis (166 mastitic quarters) caused by S. aureus treated by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine during routine farm calls were included. Treatment was based on in vitro susceptibility testing of the bacterial isolate. Procaine penicillin G (86 cases due to β -lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to β -lactamase positive strains) was administered parenterally and intramammarily for 5 days. Efficacy of treatments was assessed 2 and 4 weeks later by physical examination, bacteriological culture, determination of CMT, somatic cell count and NAGase activity in milk. Quarters with growth of S. aureus in at least one post-treatment sample were classified as non-cured. As controls we used 41 clinical mastitis cases caused by penicillin-susceptible S. aureus isolates treated with procaine penicillin G parenterally for 5 days and 15 cases due to penicillin-resistant isolates treated with spiramycin parenterally for 5 days from the same practice area. Bacteriological cure rate after the combination treatment was 75.6% for quarters infected with penicillin-susceptible S. aureus isolates, and 29.2% for quarters infected with penicillin-resistant isolates. Cure rate for quarters treated only parenterally with procaine penicillin G was 56.1% and that for quarters treated with spiramycin 33.3%. The difference in cure rates between mastitis due to penicillin-susceptible and penicillin-resistant S. aureus was highly significant. Combined treatment was superior over systemic treatment only in the β -lactamase negative group.     

Antibiotic resistance and pathogenicity factors in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from mastitic Sahiwal cattle

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. In the present study, the distribution of mastitic MRSA and antibiotic resistance was studied in 107 strains of S. aureus isolated from milk samples from 195 infected udders. The characterizations pathogenic factors (adhesin and toxin genes) and antibiotic susceptibility of isolates were carried out using gene amplification and disc diffusion assays, respectively. A high prevalence of MRSA was observed in the tested isolates (13.1%). The isolates were also highly resistant to antibiotics, i.e. 36.4% were resistant to streptomycin, 33.6% to oxytetracycline, 29.9% to gentamicin and 26.2% each to chloramphenicol, pristinomycin and ciprofloxacin. A significant variation in the expression of pathogenic factors (Ig, coa and clf) was observed in these isolates. The overall distribution of adhesin genes ebp, fib, bbp, fnbB, cap5, cap8, map and cna in the isolates was found to be 69.1, 67.2, 6.5, 20.5, 60.7, 26.1, 81.3 and 8.4%, respectively. The presence of fib, fnbB, bbp and map genes was considerably greater in MRSA than in methicillin-susceptible S. aureus (MSSA) isolates. The proportions of toxin genes, namely, hlb, seb, sec, sed, seg and sei, in the isolates were found to be 94.3, 0.9, 8.4, 0.9, 10.2 and 49.5%, respectively. The proportions of agr genes I, II, III and IV were found to be 39.2, 27.1, 21.5 and 12.1%, respectively. A few isolates showed similar antibiotic-resistance patterns, which could be due to identical strains or the dissemination of the same strains among animals. These findings can be utilized in mastitis treatment programmes and antimicrobials strategies in organized herds.     

Rapid detection of methicillin resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates; evaluation of colorimetric Quicolor ES agar and determination of breakpoint inhibition zone diameters of cefoxitin

In this study, it was aimed to evaluate colorimetric Quicolor ES agar for the rapid detection of methicillin resistance and to determine susceptibility and resistance breakpoint zone diameters for cefoxitin by using 51 methicillin susceptible Staphylococcus aureus (MSSA) and 63 methicillin resistant S. aureus (MRSA) isolates. In the study, while oxacillin and cefoxitin results were obtained within 4–7 h (5.5 h in average) for MSSA isolates, the results of MRSA isolates were obtained within 5.5–9 h (6.6 h in average) for both antibiotics on QC ES agar. QC ES agar is an inexpensive medium for rapid detection (4–9 h) of methicillin resistance by disc diffusion method using oxacillin or cefoxitin. Additional studies for further evaluation of the efficiency of QC-ES agar in rapid determination of methicillin resistance in S. aureus may be beneficial.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Variation in specificity of the PrtP extracellular proteinases in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis>

Comparison of cell-wall-bound extracellular proteinases (CEPs) from Lactobacillus paracasei (LBP) ssp. paracasei natural isolates BGHN14, BGAR75 and BGAR76 with Lactococcus lactis (LCL) ssp. cremoris Wg2, in their action on α_S1-, β- and κ-casein was done. The CEPs of LBP strains were able to degrade α_S1- and β-caseins and their caseinolytic specificity depended on the type of buffer used. These CEPs, compared with LCL Wg2, differ in four amino acid residues in small segments predicted to be involved in substrate binding. The most striking features of this comparison are the presence of Ala instead of Ser³²⁹ and the presence of Thr instead of Asn²⁵⁶ and Ala²⁹⁹, in the subtilisin-like region of the CEP in LBP natural isolates. Additional conservative amino acid substitution Leu to Ile³⁶⁴ was found.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.