CYP broth: a tool for Yersinia pestis isolation in ancient culture collections and field samples.

We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

Supervised molecular dynamics for exploring the druggability of the SARS-CoV-2 spike protein.

The recent outbreak of the respiratory syndrome-related coronavirus (SARS-CoV-2) is stimulating an unprecedented scientific campaign to alleviate the burden of the coronavirus disease (COVID-19). One line of research has focused on targeting SARS-CoV-2 proteins fundamental for its replication by repurposing drugs approved for other diseases. The first interaction between the virus and the host cell is mediated by the spike protein on the virus surface and the human angiotensin-converting enzyme (ACE2). Small molecules able to bind the receptor-binding domain (RBD) of the spike protein and disrupt the binding to ACE2 would offer an important tool for slowing, or even preventing, the infection. Here, we screened 2421 approved small molecules in silico and validated the docking outcomes through extensive molecular dynamics simulations. Out of six drugs characterized as putative RBD binders, the cephalosporin antibiotic cefsulodin was further assessed for its effect on the binding between the RBD and ACE2, suggesting that it is important to consider the dynamic formation of the heterodimer between RBD and ACE2 when judging any potential candidate.

Distinct single-cell morphological dynamics under beta-lactam antibiotics.

The bacterial cell wall is conserved in prokaryotes, stabilizing cells against osmotic stress. Beta-lactams inhibit cell-wall synthesis and induce lysis through a bulge-mediated mechanism; however, little is known about the formation dynamics and stability of these bulges. To capture processes of different timescales, we developed an imaging platform combining automated image analysis with live-cell microscopy at high time resolution. Beta-lactam killing of Escherichia coli cells proceeded through four stages: elongation, bulge formation, bulge stagnation, and lysis. Both the cell wall and outer membrane (OM) affect the observed dynamics; damaging the cell wall with different beta-lactams and compromising OM integrity cause different modes and rates of lysis. Our results show that the bulge-formation dynamics are determined by how the cell wall is perturbed. The OM plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis and allows recovery upon drug removal.

A novel peptidoglycan binding protein crucial for PBP1A-mediated cell wall biogenesis in Vibrio cholerae.

The bacterial cell wall, which is comprised of a mesh of polysaccharide strands crosslinked via peptide bridges (peptidoglycan, PG), is critical for maintenance of cell shape and survival. PG assembly is mediated by a variety of Penicillin Binding Proteins (PBP) whose fundamental activities have been characterized in great detail; however, there is limited knowledge of the factors that modulate their activities in different environments or growth phases. In Vibrio cholerae, the cause of cholera, PG synthesis during the transition into stationary phase is primarily mediated by the bifunctional enzyme PBP1A. Here, we screened an ordered V. cholerae transposon library for mutants that are sensitive to growth inhibition by non-canonical D-amino acids (DAA), which prevent growth and maintenance of cell shape in PBP1A-deficient V. cholerae. In addition to PBP1A and its lipoprotein activator LpoA, we found that CsiV, a small periplasmic protein with no previously described function, is essential for growth in the presence of DAA. Deletion of csiV, like deletion of lpoA or the PBP1A-encoding gene mrcA, causes cells to lose their rod shape in the presence of DAA or the beta-lactam antibiotic cefsulodin, and all three mutations are synthetically lethal with deletion of mrcB, which encodes PBP1B, V. cholerae's second key bifunctional PBP. CsiV interacts with LpoA and PG but apparently not with PBP1A, supporting the hypothesis that CsiV promotes LpoA's role as an activator of PBP1A, and thereby modulates V. cholerae PG biogenesis. Finally, the requirement for CsiV in PBP1A-mediated growth of V. cholerae can be overcome either by augmenting PG synthesis or by reducing PG degradation, thereby highlighting the importance of balancing these two processes for bacterial survival.

Interactions of release factor RF3 with the translation machinery.

The bacterial release factor RF3 is a GTPase that has been implicated in multiple, incompletely understood steps of protein synthesis. This study explores the genetic interaction of RF3 with other components of the translation machinery. RF3 contributes to translation termination by recycling the class I release factors RF1 and RF2 off post-termination ribosomes. RF3 has also been implicated in dissociation of peptidyl-tRNAs from elongating ribosomes and in a post-peptidyltransferase quality control (post-PT QC) mechanism that selectively terminates ribosomes carrying erroneous peptides. A majority of the in vivo studies on RF3 have been carried out in K-12 strains of Escherichia coli which carry a partially defective RF2 protein with an Ala to Thr substitution at position 246. Here, the contribution of the K-12 specific RF2 variant to RF3 activities has been investigated. Strain reconstruction experiments in both E. coli and Salmonella enterica demonstrate that defects in termination and post-PT QC that are associated with RF3 loss, as well as phenotypes uncovered by phenotypic profiling, are all substantially ameliorated when the incompletely active K-12-specific RF2 protein is replaced by a fully active Ala246 RF2. These results indicate that RF3 loss is well tolerated in bacteria with fully active class I release factors, but that many of the previously reported phenotypes for RF3 deletion strains have been compromised by the presence of a partially defective RF2.

High-resolution dose-response screening using droplet-based microfluidics.

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 µM.

[A simple disk diffusion test to identify beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae--application of cephalexin, cefsulodin and cefaclor disks].

Currently, beta-lactamase-negative (BLN) ampicillin-resistant (AR) strains of Haemophilus influenzae are prevalent in Japan. BLNAR strains are defined by the presence of specific mutation(s) in the ftsI gene but are not phenotypically distinguishable by ampicillin (ABPC) susceptibility. In the present study, we showed that cephalexin (CEX), cefsulodin (CFS), and cefaclor (CCL) disk diffusion tests can be effectively used to identify BLNAR strains. A total of 169 clinical isolates of BLN H. influenzae, consisting of 113 of BLNAR and 56 of BLN, ampicillin-susceptible (AS), were included. All the isolates were genetically defined by detection of the TEM gene and partial sequencing of the ftsI gene. The Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution and disk diffusion tests for ABPC provided 20% and 19% false susceptible rates, respectively. Alternatively, 34 cephem agents were tested using disk diffusion. Of the agents tested, CEX, CFS, and CCL disks could effectively discriminate between BLNAR and BLNAS isolates. All the BLNAS isolates showed visible growth inhibitory zones around CEX and CFS disks, but 108 (95.6%) and 106 (93.8%) BLNAR isolates did not. The results indicated 100% predictive values (PVs) for BLNAR and PVs for BLNAS were 91.8% for CEX and 88.9% for CFS. The CLSI-based interpretations for CCL (> or =20 mm) also highly correlated with BLNAR and BLNAS, PVs for BLNAR and for BLNAS being 100% and 93.3%, respectively. With simplicity and discriminability of the test method, we recommend a CEX disk diffusion test in combination with a rapid beta-lactamase test to identify BLNAR isolates in clinical laboratories.

SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli.

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

Identification of Inhibitors of Integrin Cytoplasmic Domain Interactions With Syk.

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance.

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.

Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells.

The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.

Evaluation of bactericidal activity of cefotaxime and other beta-lactams by a novel method.

In previous studies on Streptococcus faecium we proposed that the minimum beta-lactam concentration killing 99.9% of a bacterial population within 3 hours be defined as the minimum directly bactericidal concentration (MDBC) of that drug. In the present study we first evaluated the kinetics of cellular killing by various beta-lactams as related to penicillin-binding-protein (PBP) binding in Escherichia coli DC2, a hyperpermeable mutant. We concluded that in E. coli the MDBC for beta-lactams coincides with the minimum concentration capable of saturating PBPs 1b, 2 and 3. Of the antibacterial drugs we studied, cefsulodin, mecillinam and aztreonam had a much greater affinity for one essential PBP (PBP 1b, 2 and 3, respectively) than for all others, whereas cefotaxime had close affinities for all the above PBPs. MDBC values of greater than 500, 500, greater than 50, 10 and 1.5 mg/L were obtained for cefsulodin, mecillinam, aztreonam, ampicillin and cefotaxime, respectively. On the basis of the pharmacokinetic properties of these drugs, our results indicate that mecillinam, ampicillin and cefsulodin may be bactericidal in urine but not at other body sites; aztreonam is probably bactericidal in urine and blood, but not elsewhere; and cefotaxime is bactericidal in all the biological fluids we studied.

In vitro inhibitory and bactericidal activity of cefpiramide and seven antipseudomonal agents against Pseudomonas aeruginosa.

Cefpiramide was tested against 493 clinical isolates of Pseudomonas aeruginosa and the results of minimal inhibitory concentration, minimal bactericidal concentration, bactericidal rate, and time-kill synergy studies were compared with those obtained with seven other antipseudomonal agents. The minimal inhibitory concentrations of cefpiramide for P. aeruginosa were comparable to all of the agents tested. Minimal bactericidal concentration results were generally within one twofold dilution of the minimal inhibitory concentration values for all agents tested. Bactericidal rate studies showed that at concentrations of four times the minimal inhibitory concentration, all of the agents produced rapid killing. Results of time-kill synergy studies showed a marked synergistic interaction between cefpiramide and each of three aminoglycosides, gentamicin, tobramycin, and amikacin. These results suggest that cefpiramide may be useful in the therapy of infections due to P. aeruginosa.

Temperature-dependent expression of the chromosomal beta-lactamase gene in a strain of Pseudomonas aeruginosa.

A strain of Pseudomonas aeruginosa (3-Post) was resistant to cefsulodin and ceftazidime at 37 degrees C but sensitive at 20 degrees C. Resistance was mediated by chromosomally-encoded beta lactamase which was synthesised at a high level during growth above 30 degrees C but at a low, inducible level during growth below 27 degrees C.

In vitro activity of cefsulodin against multi-resistant isolates of Pseudomonas aeruginosa.

Clinical isolates of Pseudomonas aeruginosa from 245 patients with different infections were tested to determine their in vitro susceptibility to cefsulodin and other anti-pseudomonad antibiotics. Cefsulodin inhibited 90% of the isolates as compared with 89% by ceftazidime, 84% by piperacillin and 73% by ticarcillin. Of the three aminoglycosides used, gentamycin (60%) and netilmycin (77%) were less inhibitory. Amikacin was the most active, inhibiting 92% of the clinical isolates. Over 60% of the isolates resistant to ticarcillin and piperacillin were susceptible to cephalosporin and aminoglycosides used. Among cefsulodin-resistant isolates, ceftazidime was active against 67% and amikacin and netilmycin against 71% of isolates. Sixty percent of ceftazidime-resistant strains were susceptible to cefsulodin.

Effects of five antibiotics on adhesion and haemagglutinating properties of Pseudomonas aeruginosa isolated from cystic fibrosis patients.

The aim of this study was to evaluate the in vitro effect of five antibiotics at sub-inhibitory concentrations on the adhesive and haemagglutinating properties of Pseudomonas aeruginosa isolated from cystic fibrosis sputa. Eleven isolates (mucoid and non-mucoid) from cystic fibrosis, and four isolates (mucoid and non-mucoid) from other chronic respiratory infections were tested. The adhesion test was performed on human lymphoblastoid cell-lines; the haemagglutination test used human O+ and guinea-pig erythrocytes. The antibiotics were tested at six sub-inhibitory concentrations, from MIC/2 to MIC/64. Among the five antibiotics, cefsulodin and pefloxacin were the most active in decreasing the adhesive properties: this effect was statistically significant at MIC/2 and MIC/4 for cefsulodin and at all sub-inhibitory concentrations for pefloxacin. No differences appeared between mucoid and non-mucoid strains, and no correlation was noted with their clinical origins. The three other antibiotics (ceftazidime, latamoxef and imipenem) had no significant effect on the adhesion of all the strains tested, but their effect was rather strain-dependent. This fact and the heterogeneity found in adherence and haemagglutinating activity of each strain suggest that the adhesins and the haemagglutinins of P. aeruginosa are very complex structures.

Comparison of the in vitro effects of several cephalosporins on the oxidative burst of human phagocytes.

The metabolic activity (oxygen radical formation) of human phagocytes was not substantially affected by the tested cephalosporins. Therapeutic concentrations caused only a mild suppression or immunopotentiation in some cases or there were no effects altogether.

[Value of bactericidal curves in the optimal selection of a beta-lactam-aminoglycoside combination on Pseudomonas aeruginosa. In vitro study of 40 strains of Pseudomonas aeruginosa isolated in pediatric intensive care units]. / Intérêt des courbes de bactéricidie dans le choix optimal d'une association beta-lactamine-aminoside sur Pseudomonas aeruginosa. Etude in vitro de 40 souches de Pseudomonas aeruginosa isolées en réanimation infantile.

Antimicrobial sensitivity and time-kill curves were determined for ticarcillin, azlocillin, piperacillin, cefsulodin, ceftazidime, gentamicin, tobramycin and amikacin alone or in combination against 40 strains of Pseudomonas aeruginosa isolated from blood cultures and tracheal aspirates in pediatric intensive care units. The antibiotics were used in concentrations obtainable with the usual therapeutic dosage. No bactericidal effect was observed with each of the beta-lactam antibiotics tested alone. For ticarcillin-sensitive strains the most rapid bacterial inoculum size decrease was observed at 2.5 h with the piperacillin-amikacin combination, and a bactericidal effect was obtained within 4.5 h when amikacin was combined with ticarcillin, azlocillin, piperacillin, ceftazidime or cefsulodin. For ticarcillin-resistant strains a bactericidal effect was obtained within 4.5 h when amikacin was combined with piperacillin, azlocillin, ceftazidime or cefsulodin.

[In vitro combination effects of astromicin and beta-lactam antibiotics against CFS-sensitive and CFS-resistant Pseudomonas aeruginosa. In vitro synergistic activity].

We investigated in vitro synergistic activity of astromicin (ASTM) combined with beta-lactam antibiotics (cefsulodin (CFS), cefoperazone (CPZ), ceftazidime (CAZ), piperacillin (PIPC) and fosfomycin (FOM) against fresh clinical isolated Pseudomonas aeruginosa, which consisted of 13 CFS sensitive (MIC less than or equal to 3.13 micrograms/ml) and 19 CFS resistant (MIC greater than or equal to 400 micrograms/ml) strains according to the FIC index. Against CFS-sensitive P. aeruginosa, ASTM showed good synergistic activities when combined with PIPC (54%), CAZ (38%), CPZ (23%) and CFS (8%). Against CFS-resistant P. aeruginosa, ASTM also showed high synergistic activities when combined with CAZ (63%), CPZ (47%), PIPC (37%) and CFS (11%). Among the CFS-resistant P. aeruginosa, one clinical isolate showed a high sensitivity (MIC0.78 micrograms/ml) against ASTM alone.

[Susceptibility of clinical isolates to aztreonam].

Aztreonam (AZT), which has a newly developed and synthetic structure belonging to the group of monobactams, possesses excellent antibacterial activity against a broad range of Gram-negative bacteria (including the beta-lactamase producing strains). Antibacterial activities of AZT were examined and compared with those of 5 antibiotics (cefoperazone (CPZ), latamoxef (LMOX), cefotaxime (CTX), cefmetazole (CMZ) and cefsulodin (CFS) against 296 strains of clinical isolates. The evaluation of antibacterial activities was determined with the inhibition zone diameter obtained by the single disc method. The results can be summarized by three categories as follows: 1. Susceptibility of clinical isolates to AZT and other antibiotics AZT and other 3rd-generation antibiotics (CPZ, LMOX, CTX) showed excellent antibacterial activities against most strains. Especially AZT was more active against Enterobacter cloacae and Serratia marcescens than reference drugs. Against Pseudomonas aeruginosa, the activity of AZT was similar to that of CFS. AZT showed as excellent activity against P. aeruginosa as CFS. 2. Susceptibility of strains isolated from different clinical materials and different clinics AZT showed the highest antibacterial activity against the clinical isolates from sputum, pharynx, urine, pus, bile, puncture fluid, blood and others. AZT exhibited the most potent activity against isolates in the 7 clinics we tested. 3. Susceptibility of strains isolated from inpatients and outpatients AZT demonstrated the highest antibacterial activity (the rate of susceptibility: 87.0%) against strains obtained from inpatients (except for P. aeruginosa). The activity of AZT (81.4%) against P. aeruginosa was as active as that of CFS (81.4%), and it was the highest in all drugs. Antibacterial activity of these antibiotics against bacteria was rated as follows: AZT greater than LMOX greater than CPZ greater than CTX greater than CMZ AZT showed the highest antibacterial activity (100%) against strains isolated in all the materials and at all the clinics tested of outpatients. Antibacterial activity of these antibiotics against isolates from outpatients was rated as follows: AZT greater than CPZ greater than LMOX greater than CTX greater than CMZ.