Self-Competitive Inhibition of the Bacteriophage P22 Tailspike Endorhamnosidase by O-Antigen Oligosaccharides.

The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of O-antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be ∼7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.

Elucidation of the O-antigen structure of Escherichia coli O63.

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-ß-d-Quip3N(d-allo-ThrAc)-(1→2)-ß-d-Ribf-(1→4)-ß-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element ß-d-Galp-(1→3)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.

Structure and gene cluster of the O-antigen of Escherichia coli O165 containing 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid.

Upon mild acid degradation of the lipopolysaccharide of Escherichia coli O165, the O-polysaccharide chain was cleaved at the glycosidic linkage of 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Hb7Ac). Analysis of the resulting linear tetrasaccharide and alkali-treated lipopolysaccharide by (1)H/(13)C 1D and 2D nuclear magnetic resonance spectroscopy enabled elucidation of the following structure of the O-polysaccharide: →8)-α-Psep5Hb7Ac-(2 → 6)-ß-d-Galp-(1 → 4)-ß-d-Glсp-(1 → 3)-α-d-GlсpNAc-(1→. The ß-d-Galp-(1 → 4)-ß-d-Glсp-(1 → 3)-d-GlсpNAc structural element is also present in the O-polysaccharide of E. coli O82. The content of the O-antigen gene cluster of E. coli O165 was found to be consistent with the O-polysaccharide structure established. Functions of proteins encoded in the gene cluster, including enzymes involved in the Pse5Hb7Ac biosynthesis and glycosyltransferases, were putatively assigned by comparison with sequences in available databases.

Ecological Effect of Solithromycin on Normal Human Oropharyngeal and Intestinal Microbiota.

Solithromycin is a new fluoroketolide. The purpose of the present study was to investigate the effect of orally administered solithromycin on the human oropharyngeal and intestinal microbiota. Thirteen healthy volunteers (median age, 27.3 years) received oral solithromycin at 800 mg on day 1 followed by 400 mg daily on days 2 to 7. Fecal and saliva samples were collected at baseline and on days 2, 5, 7, 9, 14, and 21 for pharmacokinetic and microbiological analyses. Plasma samples were collected predose on days 2, 5, and 7 as proof of exposure, and solithromycin concentration ranges were 21.9 to 258 ng/ml, 18.0 to 386 ng/ml, and 16.9 to 417 ng/ml, respectively. The solithromycin concentrations in feces were 15.8 to 65.4 mg/kg, 24.5 to 82.7 mg/kg, 21.4 to 82.7 mg/kg, 12.1 to 72.4 mg/kg, 0.2 to 25.6 mg/kg, and 0 to 0.5 mg/kg on days 2, 5, 7, 9, 14, and 21, respectively. The numbers of enterobacteria and enterococci decreased and were normalized on day 14. The numbers of lactobacilli and bifidobacteria decreased from day 2 to day 14 and were normalized on day 21. The clostridia decreased on days 2, 7, and 14 and were normalized on day 21. No Clostridium difficile strains or toxins were detected during the study period. The number of Bacteroides strains was not significantly changed. The solithromycin concentrations in saliva were 0 to 1.2 mg/liter, 0 to 0.5 mg/liter, 0 to 0.5 mg/liter, and 0 to 0.1 mg/liter on days 2, 5, 7, and 9, respectively. The numbers of streptococci decreased on day 2 and were normalized on day 5. The numbers of lactobacilli, prevotellae, fusobacteria, and leptotrichiae decreased from day 2 and were normalized on day 21.

In-vitro activity of solithromycin against anaerobic bacteria from the normal intestinal microbiota.

Solithromycin is a novel fluoroketolide with high activity against bacteria associated with community-acquired respiratory tract infections as well as gonorrhea. However, data on the activity of solithromycin against anaerobic bacteria from the normal intestinal microbiota are scarce. In this study, 1024 Gram-positive and Gram-negative anaerobic isolates from the normal intestinal microbiota were analyzed for in-vitro susceptibility against solithromycin and compared to azithromycin, amoxicillin/clavulanic acid, ceftriaxone, metronidazole and levofloxacin by determining the minimum inhibitory concentration (MIC). Solithromycin was active against Bifidobacteria (MIC50, 0.008 mg/L) and Lactobacilli (MIC50, 0.008 mg/L). The MIC50 for Clostridia, Bacteroides, Prevotella and Veillonella were 0.5, 0.5, 0.125 and 0.016 mg/L, respectively. Gram-positive anaerobes were more susceptible to solithromycin as compared to the other antimicrobials tested. The activity of solithromycin against Gram-negative anaerobes was equal or higher as compared to other tested agents.

Clostridium difficile recurrences in Stockholm.

Sixty-eight hospital-admitted patients with a first episode of Clostridium difficile infection (CDI) were included and followed up during 1 year. Faeces samples were collected at 1, 2, 6 and 12 months after inclusion and analyzed for the presence of C. difficile toxin B, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped and the MICs of the isolates were determined against eight antimicrobial agents. In 68 patients initially included, antibiotics, clinical signs and co-morbidities were analyzed and 56 were evaluable for recurrences. The mean number of different antibiotics given during 3 months prior to inclusion was 2.6 (range 0-6). Six patients had not received any antibiotics and three of them had diagnosed inflammatory bowel disease. Thirty-two patients (57%) had either a microbiological or clinical recurrence, 16 of whom had clinical recurrences that were confirmed microbiologically (13, 23%) or unconfirmed by culture (3, 5%). Twenty-nine patients were positive in at least one of the follow-up tests, 16 had the same ribotype in follow-up tests, i.e. relapse, and 13 a different ribotype, i.e., reinfection. Most common ribotypes were 078/126, 020, 023, 026, 014/077, 001 and 005. No strain of ribotype 027 was found. Strains ribotype 078/126 and 023 were positive for binary toxin and were the strains most prone to cause recurrence. All strains were sensitive to vancomycin and metronidazole. Patients with recurrences were significantly older (p = 0.02) and all patients had a high burden of comorbidities, which could explain the high fatality rate, 26 (38%) patients died during the 1-year follow-up.

Determining the Long-term Effect of Antibiotic Administration on the Human Normal Intestinal Microbiota Using Culture and Pyrosequencing Methods.

The purpose of the study was to assess the effect of ciprofloxacin (500 mg twice daily for 10 days) or clindamycin (150 mg 4 times daily for 10 days) on the fecal microbiota of healthy humans for a period of 1 year as compared to placebo. Two different methods, culture and microbiome analysis, were used. Fecal samples were collected for analyses at 6 time-points. The interval needed for the normal microbiota to be normalized after ciprofloxacin or clindamycin treatment differed for various bacterial species. It took 1-12 months to normalize the human microbiota after antibiotic administration, with the most pronounced effect on day 11. Exposure to ciprofloxacin or clindamycin had a strong effect on the diversity of the microbiome, and changes in microbial composition were observed until the 12th month, with the most pronounced microbial shift at month 1. No Clostridium difficile colonization or C. difficile infections were reported. Based on the pyrosequencing results, it appears that clindamycin has more impact than ciprofloxacin on the intestinal microbiota.

Ecological Effect of Ceftaroline-Avibactam on the Normal Human Intestinal Microbiota.

Ceftaroline-avibactam is a new combination of the antibiotic ceftaroline with a novel non-ß-lactam ß-lactamase inhibitor, avibactam. The purpose of the present study was to investigate the effect of ceftaroline-avibactam on the human intestinal microbiota. Fourteen healthy volunteers received ceftaroline-avibactam (600 mg ceftaroline fosamil and 600 mg avibactam) intravenously over 2 h every 8 h on days 1 to 6 and as a single dose on day 7. Fecal samples were collected on day -1 (within 24 h of the first infusion on day 1) and on days 2, 5, 7, 9, 14, and 21. Escherichia coli numbers decreased during the study and normalized on day 21. An increased number of Klebsiella bacteria appeared on day 14 and normalized on day 21. The number of other enterobacteria decreased during the study, and the number of enterococci decreased from days 2 to 7 and normalized on day 9. Candida numbers increased from days 5 to 9 and normalized after day 14. The number of lactobacilli decreased during the study and recovered on day 14. The number of bifidobacteria decreased on day 2 and normalized on day 21. The number of Bacteroides bacteria was unchanged. Clostridium difficile numbers decreased on days 7 and 9 and increased on days 14 and 21. A toxigenic C. difficile strain was detected in one volunteer on day 21 with no reported adverse events. Plasma samples were collected on days -1, 2, 5, and 7. Ceftaroline and avibactam concentrations were 0 to 34.5 mg/liter and 0 to 61.6 mg/liter, respectively, in plasma and 0 to 35.4 mg/kg and 0 to 98.5 mg/kg, respectively, in feces. (This study is registered in the European Clinical Trials Database [https://eudract.ema.europa.eu/] under number EudraCT 2012 004921-25.).

Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor.

The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum ß-lactamase blaCTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein.

Development of antimicrobial resistance in the normal anaerobic microbiota during one year after administration of clindamycin or ciprofloxacin.

Thirty healthy subjects (15 males and 15 females) were randomly assigned in three groups and clindamycin (150 mg qid) or ciprofloxacin (500 mg bid) or placebo was given for a 10-day period. Skin, nasal, saliva, faeces samples were collected at day - 1, day 11, 1 month, 2 months, 4 months and 12 months post administration for microbiological analysis. Ciprofloxacin or clindamycin had no impact on the anaerobic skin microbiota and the proportions of antibiotic resistant anaerobic bacteria were similar as in the placebo group. Ciprofloxacin had impact on the Propionibacterium acnes in the nasal microbiota that normalized after 1 month, however, ciprofloxacin-resistant P. acnes strains increased at month 2 and month 12. Clindamycin had no impact on the nasal microbiota. In the oropharyngeal microbiota, a higher proportion of ciprofloxacin resistant Veillonella was found, it lasting up to 12 months post dosing. In the clindamycin group, clindamycin-resistant Prevotella spp. were found in increased proportions compared to placebo at various time points except month 4 in the saliva samples. The relative proportion of ciprofloxacin-resistant Bifidobacteria increased in the faecal samples on day 11, 1 month, 4 months and 12 months post dosing compared to placebo. The proportion of clindamycin-resistant Bacteroides spp. increased at 1, 2, 4 and 12 months post dosing compared to placebo in the faecal samples. No Clostridium difficile was recovered from any of the samples from any of the volunteers at any visit. The concentrations of ciprofloxacin or clindamycin in the faeces were higher than the MICs for most of the organisms present in the normal microbiota. No obvious correlation between the groups in resistant patterns for anaerobic bacteria was observed. In conclusion, based on the microbiological data of the microbiota as well as the results of the bioassays for ciprofloxacin and clindamycin concentrations in the faecal samples, oral administration of ciprofloxacin and clindamycin has an impact on the anaerobic microbiota and may have a long-term effect on the development and persistence of antibiotic-resistant anaerobes in the normal microbiota.

Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: application to the O-antigen of Escherichia coli O59.

A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, →6)[α-d-GalpA3Ac/4Ac-(1 → 3)]-α-d-Manp-(1 → 3)-α-d-Manp-(1 → 3)-ß-d-Manp-(1 → 3)-α-d-GlcpNAc-(1→. The identification of the O-acetylation positions was efficiently performed by comparison of the (1)H,(13)C HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

Alternative strategies for proof-of-principle studies of antibacterial agents.

The proof that a new antibacterial agent is not only active in vitro but also effective in vivo under clinically relevant conditions is currently provided (i) by using appropriate nonclinical models of infection and pharmacokinetic-pharmacodynamic (PK-PD) analysis providing evidence of the likelihood of clinical efficacy and (ii) by examining the study drug in exploratory clinical trials, as well as dose and schedule finding during phase II of clinical development. This approach is both time-consuming and costly. Furthermore, PK-PD targets for any novel antibacterial agent cannot be derived from studies with experimental animals. Therefore, alternative strategies have to be identified to prove the principle that a novel antibacterial agent is active under clinically relevant conditions. This review summarizes evidence that the quantitative analysis of shifts in the viable counts of pathogens in infected patients or the evaluation of the PD effect of an investigational agent on indicator organisms of the human resident microflora or colonizers of healthy volunteers, if paralleled with PK monitoring of serum and the target site, provides an alternative to a classical proof-of-principle study in the course of a phase II study program.

Toxin-producing Clostridium difficile strains as long-term gut colonizers in healthy infants.

Clostridium difficile is a colonizer of the human gut, and toxin-producing strains may cause diarrhea if the infectious burden is heavy. Infants are more frequently colonized than adults, but they rarely develop C. difficile disease. It is not known whether strains of C. difficile differ in the capacity to colonize and persist in the human gut microbiota. Here, we strain typed isolates of C. difficile that had colonized 42 healthy infants followed from birth to ≥12 months of age by using PCR ribotyping of the 16S-23S rRNA intergenic spacer region. The isolates were also characterized regarding carriage of the toxin genes tcdA, tcdB, and cdtA/B and the capacity to produce toxin B in vitro. Most strains (71%) were toxin producers, and 51% belonged to the 001 or 014 ribotypes, which often cause disease in adults. These ribotypes were significantly more likely than others to persist for ≥6 months in the infant micobiota, and they were isolated from 13/15 children carrying such long-term-colonizing strains. Ribotype 001 strains were often acquired in the first week of life and attained higher population counts than other C. difficile ribotypes in newborn infants' feces. Several toxin-negative ribotypes were identified, two of which (GI and GIII) were long-term colonizers, each found in one infant. Our results suggest that the toxin-producing C. difficile ribotypes 001 and 014 have special fitness in the infantile gut microbiota. Toxin-producing strains colonizing young children for long time periods may represent a reservoir for strains causing disease in adults.

In vitro activity of MCB3681 against Clostridium difficile strains.

One hundred fourteen Clostridium difficile strains were collected from 67 patients and analyzed for the presence of C. difficile toxin B by the cell cytotoxoicity neutralization assay, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped. The MICs of the isolates were determined against MCB3681 and nine other antimicrobial agents by the agar dilution method. All isolates were positive for toxin B as well as for toxin A and B genes. In addition, 13 isolates were positive for the binary toxin genes. Thirty-two different ribotypes were identified. No strain of ribotype 027 was found. All 114 isolates were sensitive to MCB3681 (0.008-0.5 mg/l), cadazolid (0.064-0.5 mg/l), fidaxomicin (0.008-0.125 mg/l), metronidazole (0.125-2 mg/l), vancomycin (0.125-1 mg/l) and tigecycline (0.032-0.25 mg/l). Three isolates were resistant to linezolid (8 mg/l), 12 isolates were resistant to moxifloxacin (8-32 mg/l), 87 isolates were resistant to clindamycin (8-256 mg/l) and 107 isolates were resistant to ciprofloxacin (8-256 mg/l). No association between toxins A, B and binary toxin, ribotypes and the sensitivity to MCB3681 could be found. MCB3681 has a potent in vitro activity against C. difficile.

Risk factors of Clostridium difficile infections among patients in a university hospital in Shanghai, China.

Clostridium difficile infection (CDI) is an increasing concern in China. However, the risk factors of CDI are rarely reported in the Chinese population. A prospective observational study was therefore conducted among patients with hospital-acquired C. difficile diarrhoea and the risk factors of CDI in a retrospective case-control study. The CDI patients were compared with the non-CDI diarrhoeal patients and those without diarrhoea, respectively. The recurrent CDI patients were compared with the corresponding non-recurrent CDI patients and those without diarrhoea, respectively. Overall, of the 240 patients with hospital-acquired diarrhoea 90 (37.5%) were diagnosed as CDI, and 12 (13.3%) of the 90 CDI patients experienced recurrence. Multivariate analysis indicated that renal disease, malignancy, hypoalbuminemia, prior antibiotic treatment, chemotherapy, nasogastric tube use, length of stay>14 days and intra-abdominal surgery, defined daily dose of antimicrobial agents≥19, prior use of more than three antimicrobial agents, and use of carbapenems were independent risk factors for the first episode of CDI. Use of laxatives, the first- and second-generation narrow-spectrum cephalosporins or metronidazole was identified as protective factors. It is necessary to make testing of C. difficile available as a routine practice and control these risk factors in Chinese hospitals to avoid CDI outbreaks.

Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof.

The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[ß-L-Rhap-(1 → 4)]-ß-D-GlcpNAc-(1 → 4)-α-D-GalpA-(1 → 3)-α-D-Manp-(1 → 3)-ß-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a ß-L-Rhap-(1 → 4)-ß-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-D-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of the l-Rhap residue (∼0.70 per repeating unit) as well as at O-2 and O-3 of the D-GalpA residue (∼0.03 and ∼0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286-290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.

In vitro activity of cadazolid against Clostridium difficile strains isolated from primary and recurrent infections in Stockholm, Sweden.

One hundred thirty-three Clostridium difficile strains were collected from 71 patients and analyzed for the presence of C. difficile toxin B by the cell cytotoxicity neutralization assay, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped and analyzed for sporulation frequency. The MICs of the isolates were determined against cadazolid and seven other antimicrobial agents by the agar dilution method. All isolates were positive for toxin B by the cell cytotoxicity neutralization assay. One hundred fourteen isolates were positive for toxin A and B and 16 isolates were positive for toxin A, toxin B and binary toxin by PCR. Three isolates were negative for toxin A but positive for toxin B. Thirty-three different ribotypes were identified. No strain of ribotype 027 was found. No differences in sporulation were noticed between the primary and recurrent isolates. All 133 isolates were sensitive to cadazolid (0.064-0.5 mg/l), fidaxomicin (0.008-0.125 mg/l), metronidazole (0.125-2 mg/l), vancomycin (0.125-1 mg/l) and tigecycline (0.032-0.25 mg/l). Three isolates were resistant to linezolid (8 mg/l), 15 isolates were resistant to moxifloxacin (8-32 mg/l) and 103 isolates were resistant to clindamycin (8-256 mg/l). No association between toxins A, B and binary toxin, ribotypes or the sporulation and the sensitivity to cadazolid could be found. Cadazolid has a potent in vitro activity against C. difficile.

Effect of new antimicrobial agents on the ecological balance of human microflora.

The human normal microflora is relatively stable at each ecological habitat under normal circumstances and acts as a barrier against colonization by potentially pathogenic microorganisms and against overgrowth of already present opportunistic microorganisms. Administration of antimicrobial agents causes disturbances in the ecological balance between the host and the normal microflora. The risk of emergence and spread of resistant strains between patients and dissemination of resistant determinants between microorganisms is reduced if colonization resistance is not disturbed by antimicrobial agents. In this article, the potential ecological effects of administration of new antimicrobial agents on the intestinal and oropharyngeal microflora are summarized. The review is based on clinical studies published during the past 10 years.

Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative.

O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.

Effect of ceftaroline on normal human intestinal microflora.

Ceftaroline is a new broad-spectrum cephalosporin being developed for the treatment of serious bacterial infections, including those caused by aerobic Gram-positive and Gram-negative bacteria. The purpose of the present study was to investigate the effect of administration of ceftaroline on the intestinal flora of healthy subjects. Twelve healthy subjects (6 males and 6 females), 20 to 41 years of age, received ceftaroline (600 mg) by intravenous infusion every 12 h (q12h) for 7 days. Plasma and feces were collected for determination of ceftaroline concentration and analysis of fecal flora. Fecal specimens were cultured on nonselective and selective media. Different colony types were counted, isolated in pure culture, and identified to the genus level. All new strains of colonizing bacteria were tested for susceptibility to ceftaroline. The concentrations of ceftaroline in plasma were as follows: on day 2, 17.5 to 34.8 mg/liter; on day 5, 19.7 to 33.2 mg/liter; and on day 7, 18.0 to 29.8 mg/liter. No ceftaroline concentrations were found on day -1, 9, 14, or 21. No measurable concentrations in feces were found on day -1, 2, 5, 7, 9, 14, or 21. There was a minor impact on the numbers of Escherichia coli strains, while the numbers of enterococci and Candida albicans strains were not affected. There were moderate decreases in the numbers of bifidobacteria and lactobacilli during the first 7 days, while the numbers of clostridia increased during the same period. No impact on the numbers of Bacteroides bacteria was noticed. No new colonizing aerobic or anaerobic bacteria resistant to ceftaroline (MIC >or= 4 mg/liter) were found. Ceftaroline had no significant ecological impact on the human intestinal microflora.