Preclinical Immunogenicity and Efficacy of Optimized O25b O-Antigen Glycoconjugates To Prevent MDR ST131 E. coli Infections.

Multivalent O-antigen polysaccharide glycoconjugate vaccines are under development to prevent invasive infections caused by pathogenic Enterobacteriaceae. Sequence type 131 (ST131) Escherichia coli of serotype O25b has emerged as the predominant lineage causing invasive multidrug-resistant extraintestinal pathogenic E. coli (ExPEC) infections. We observed the prevalence of E. coli O25b ST131 among a contemporary collection of isolates from U.S. bloodstream infections from 2013 to 2016 (n = 444) and global urinary tract infections from 2014 to 2017 (n = 102) to be 25% and 24%, respectively. To maximize immunogenicity of the serotype O25b O antigen, we investigated glycoconjugate properties, including CRM197 carrier protein cross-linking (single-end versus cross-linked "lattice") and conjugation chemistry (reductive amination chemistry in dimethyl sulfoxide [RAC/DMSO] versus ((2-((2-oxoethyl)thio)ethyl)carbamate [eTEC] linker). Using opsonophagocytic assays (OPAs) to measure serum functional antibody responses to vaccination, we observed that higher-molecular-mass O25b long-chain lattice conjugates showed improved immunogenicity in mice compared with long- or short-chain O antigens conjugated via single-end attachment. The lattice conjugates protected mice from lethal challenge with acapsular O25b ST131 strains as well as against hypervirulent O25b isolates expressing K5 or K100 capsular polysaccharides. A single 1-µg dose of long-chain O25b lattice conjugate constructed with both chemistries also elicited robust serum IgG and OPA responses in cynomolgus macaques. Our findings show that key properties of the O-antigen carrier protein conjugate such as saccharide epitope density and degree of intermolecular cross-linking can significantly enhance functional immunogenicity.

Ribotype Classification of Clostridioides difficile Isolates Is Not Predictive of the Amino Acid Sequence Diversity of the Toxin Virulence Factors TcdA and TcdB.

Clostridioides (Clostridium) difficile is the most commonly recognized cause of infectious diarrhea in healthcare settings. Currently there is no vaccine to prevent initial or recurrent C. difficile infection (CDI). Two large clostridial toxins, TcdA and TcdB, are the primary virulence factors for CDI. Immunological approaches to prevent CDI include antibody-mediated neutralization of the cytotoxicity of these toxins. An understanding of the sequence diversity of the two toxins expressed by disease causing isolates is critical for the interpretation of the immune response to the toxins. In this study, we determined the whole genome sequence (WGS) of 478 C. difficile isolates collected in 12 countries between 2004 and 2018 to probe toxin variant diversity. A total of 44 unique TcdA variants and 37 unique TcdB variants were identified. The amino acid sequence conservation among the TcdA variants (≥98%) is considerably greater than among the TcdB variants (as low as 86.1%), suggesting that different selection pressures may have contributed to the evolution of the two toxins. Phylogenomic analysis of the WGS data demonstrate that isolates grouped together based on ribotype or MLST code for multiple different toxin variants. These findings illustrate the importance of determining not only the ribotype but also the toxin sequence when evaluating strain coverage using vaccine strategies that target these virulence factors. We recommend that toxin variant type and sequence type (ST), be used together with ribotype data to provide a more comprehensive strain classification scheme for C. difficile surveillance during vaccine development objectives.

S. aureus colonization in healthy Australian adults receiving an investigational S. aureus 3-antigen vaccine.

OBJECTIVES: Assess Staphylococcus aureus (S. aureus) colonization in healthy Australian adults receiving an investigational S. aureus 3-antigen vaccine (SA3Ag). METHODS: In this phase 1, double-blind, sponsor-unblinded study, participants were randomized to receive a single dose (1 of 3 dose levels) of SA3Ag or placebo and a booster dose or placebo at 6 months. S. aureus isolates from nasal, perineal, and oropharyngeal swabs before and through 12 months post-vaccination were identified. RESULTS: Baseline S. aureus colonization prevalence was 30.6% (any site), with nasal carriage (27.0%) more common than oropharyngeal/perineal (3.2% each). Following initial vaccination (low-dose: 102; mid-dose: 101; high-dose: 101; placebo: 102) and booster (low-dose: 45; mid-dose: 44; high-dose: 27; placebo: 181), placebo and SA3Ag groups showed similar S. aureus carriage through 12 months. Most colonized participants (74.0%) were colonized by single spa types. Placebo and SA3Ag groups had similar persistence of colonization, with 19.6-30.7% due to single spa types. Acquisition was observed in mid- and high-dose recipients (∼20%) and low-dose and placebo recipients (∼12%). Vaccination resulted in substantial increases in antibodies to all 3 antigens, irrespective of carriage status. CONCLUSIONS: Based on descriptive analyses of this small study, SA3Ag vaccination did not impact S. aureus acquisition or carriage. Carriage status did not impact antibody responses to SA3Ag.

Molecular epidemiology and expression of capsular polysaccharides in Staphylococcus aureus clinical isolates in the United States.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009-10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention.

A Longitudinal Epidemiology Study of Meningococcal Carriage in Students 13 to 25 Years Old in Quebec.

Neisseria meningitidis carriage data are necessary to inform serogroup B (NmB) immunization program implementation. This longitudinal study compared detection methods to measure N. meningitidis throat carriage prevalence in Quebec from November 2010 to December 2013 using cultured swab isolates and direct swab PCR from students in ninth grade (aged 13 to 15 years; n = 534) and eleventh grade/college entry (16 to 18 years; n = 363) and in university students in dormitories (18 to 25 years; n = 360) at 3 time points per group. Meningococcal and NmB carriage rates were lower in ninth- and eleventh-grade/college entry students than university students, regardless of methodology. Genotyping cultured isolates by PCR detected NmB and non-NmB in 2.1% and 7.3% of ninth-grade students, in 1.7% and 7.2% of eleventh-grade/college entry students, and in 7.5% and 21.9% of university students, respectively. NmB acquisition rates were 1.9, 0.7, and 3.3 per 1,000 person-months across respective age groups. Most NmB isolates (94.7%, 76.9%, and 86.8%, respectively) expressed subfamily A factor H binding-protein (fHBP) variants. The most common non-NmB serogroups were NmY (1.7%/1.1%) from ninth grade and eleventh grade/college entry and NmW (2.8%) from university students. Genomic analyses detected disease-associated sequence types in carriage isolates, and carriage could persist for months. This is the largest longitudinal carriage study in Canada and the first to report fHBP variants in NmB carriage isolates in healthy Canadians. These data contribute to identification of the optimal window for NmB vaccination in precollege adolescents and provide a baseline for investigating NmB vaccination effects on carriage in this population.IMPORTANCE Disease caused by Neisseria meningitidis is associated with serious complications and a high fatality rate. Asymptomatic individuals can harbor the bacterium in the throat, a state known as "carriage," which can lead to person-to-person spread of the pathogen. This study examined N. meningitidis carriage from 2010 to 2013 among 2 groups in the Quebec City region: ninth-grade students (aged 13 to 15 years), who were also followed in their last year of high school (eleventh grade/college entry; 16 to 18 years), and university students (18 to 25 years); both groups have been shown in some other geographic regions to have high rates of carriage. This study demonstrated that N. meningitidis carriage rates were higher among university students in dormitories than ninth-grade and eleventh-grade/college entry students. Understanding carriage rates in these age groups leads to better strategies to control N. meningitidis by targeting vaccination to those responsible for transmission within the population.

Comparison of Phenotypic and Genotypic Approaches to Capsule Typing of Neisseria meningitidis by Use of Invasive and Carriage Isolate Collections.

Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitis; however, MnB is most commonly associated with asymptomatic carriage in the nasopharyngeal cavity, as opposed to the disease state. Two vaccines are now licensed for the prevention of MnB disease; a possible additional benefit of these vaccines could be to protect against disease indirectly by disrupting nasopharyngeal carriage (e.g., herd protection). To investigate this possibility, accurate diagnostic approaches to characterize MnB carriage isolates are required. In contrast to invasive meningococcal disease (IMD) isolates, which can be readily serogrouped, carriage isolates often lack capsule expression, making standard phenotypic assays unsuitable for strain characterization. Several antibody-based methods were evaluated for their abilities to serogroup isolates and were compared with two genotyping methods (real-time PCR [rt-PCR] and whole-genome sequencing [WGS]) to identify which approach would most accurately ascertain the polysaccharide groups associated with carriage isolates. WGS and rt-PCR were in agreement for 99% of IMD isolates, including those with coding sequences for MnB, MnC, MnW, and MnY, and the phenotypic methods correctly identified serogroups for 69 to 98% of IMD isolates. In contrast, only 47% of carriage isolates were groupable by genotypic methods, due to mutations within the capsule operon; of the isolates identified by genotypic methods, ≤43% were serogroupable with any of the phenotypic methods tested. These observations highlight the difficulties in the serogrouping and capsular genogrouping of meningococcal carriage isolates. Based on our findings, WGS is the most suitable approach for the characterization of meningococcal carriage isolates.

Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

In vitro activity of tigecycline against patient isolates collected during phase 3 clinical trials for diabetic foot infections.

The in vitro activity of tigecycline and comparative antimicrobial agents was evaluated against 1828 primary baseline pathogens isolated from 844 patients enrolled in the phase 3 clinical trials investigating the efficacy of tigecycline in diabetic foot infection (DFI). The trials were global, enrolling patients in 30 countries. Tigecycline was active against the most prevalent pathogens in DFI, including Gram-positive and Gram-negative isolates of both aerobic and anaerobic bacteria with 95% of MICs < or =2 microg/mL for the entire collection. The spectrum of activity of tigecycline included important pathogens for DFI, such as Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Bacteroides fragilis. As reported previously, Pseudomonas aeruginosa and several pathogens in the Proteeae group were generally less susceptible to tigecycline by comparison to other Gram-negative pathogens. The excellent in vitro expanded broad-spectrum activity of tigecycline in the clinical isolates confirmed the potential utility of tigecycline for pathogens associated with DFIs.

In vitro activity of tigecycline against patient isolates collected during phase 3 clinical trials for hospital acquired pneumonia.

The in vitro activity of tigecycline was evaluated against 819 baseline pathogens isolated from 383 patients enrolled in the phase 3 clinical trial investigating the efficacy of tigecycline in hospital acquired pneumonia (HAP). The trials were global, enrolling patients in 27 countries. Tigecycline was active against the most prevalent pathogens in HAP, including gram-positive and gram-negative strains (90% of MICs ≤2 µg/mL for the entire collection). The spectrum of activity of tigecycline included important pathogens such as Staphylococcus aureus (including methicillin-resistant S. aureus), Enterococcus faecalis, Streptococcus pneumoniae, Acinetobacter baumannii/calcoaceticus complex, Escherichia coli, Klebsiella pneumonia, and Enterobacter cloacae. As reported previously, a few genera, such as Pseudomonas aeruginosa and the Proteeae, were generally less susceptible to tigecycline by comparison to other gram-negative pathogens. The excellent in vitro, expanded, broad-spectrum activity of tigecycline in the clinical isolates confirmed the potential utility of tigecycline for pathogens associated with with hospital acquired pneumonia infections.

Resistance development profiling of piperacillin in combination with the novel {beta}-lactamase inhibitor BLI-489.

OBJECTIVES: To evaluate development of resistance to the piperacillin/BLI-489 combination. METHODS: BLI-489 was used at a constant concentration of 4 mg/L. Spontaneous mutation frequency was measured on piperacillin/BLI-489-containing agar plates. Five beta-lactamase-producing strains were exposed to a serial dilution of piperacillin/BLI-489, and the highest concentration allowing growth was used to inoculate subsequent serial passage for 10 days. Mutation stability was monitored in drug-free medium for 10 days. RESULTS: Escherichia coli (OXA-3, OXA-7, ACT-1, SHV-1 or none), Salmonella enterica serovar Typhimurium (CTX-M-5), Klebsiella pneumoniae (SHV-1 and SHV-5) and Enterobacter cloacae (AmpC) had a spontaneous mutation frequency of < or =1.0 x 10(-9). Two AmpC-producing Pseudomonas aeruginosa strains had a mutation frequency of 6.52 x 10(-6) and 1.0 x 10(-7); a beta-lactamase-negative P. aeruginosa strain had a mutation frequency of 2.68 x 10(-8). The mutant prevention concentration (MPC) was < or =32 mg/L. During serial passages, the MIC increased 64- and 128-fold for S. enterica serovar Typhimurium (CTX-M-5) and E. cloacae (AmpC), respectively, to > or =512 mg/L. The MIC reverted to < or =64 mg/L after serial passages in drug-free medium. The MICs increased only 4-fold for K. pneumoniae (SHV-1 and SHV-5), E. coli (OXA-3) and E. coli (SHV-1). CONCLUSIONS: Piperacillin/BLI-489 demonstrated a low probability of spontaneous resistance development in vitro for all of the strains tested with the exception of P. aeruginosa. The MPC value for all strains was < or =32 mg/L. Resistance developed during serial passage for two of the five strains tested; however, this resistance phenotype was unstable as MIC values reverted to < or =64 mg/L after propagation in drug-free medium.

Efficacy of piperacillin combined with the Penem beta-lactamase inhibitor BLI-489 in murine models of systemic infection.

The in vivo efficacy of piperacillin in combination with the penem inhibitor BLI-489 was determined using acute lethal systemic infections in mice. On the basis of preliminary results with various ratios, a dosing ratio of 8:1 was found to be optimal for retention of enhanced efficacy. Piperacillin-BLI-489 dosed at an 8:1 ratio was efficacious against murine infections caused by class A (including extended-spectrum beta-lactamases), class C (AmpC), and class D beta-lactamase-expressing pathogens.

Characterization and sequence analysis of extended-spectrum-{beta}-lactamase-encoding genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during tigecycline phase 3 clinical trials.

In concert with the development of novel beta-lactams and broad-spectrum cephalosporins, bacterially encoded beta-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-beta-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla(TEM), bla(SHV), bla(OXA), and bla(CTX) genes from clinical strains. Isolates were also screened for AmpC genes of the bla(CMY), bla(ACT), bla(FOX), and bla(DHA) families as well as the bla(KPC) genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of > or =2 microg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla(CTX-M)-type beta-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla(SHV) genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC(90) = 2 microg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.

Pyrosequencing using the single-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, and blaTEM-155.

TEM- and SHV-type extended-spectrum beta-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the bla(TEM) and bla(SHV) genes. Three novel beta-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing bla(SHV-105) were >128, 128, and >128 microg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla(TEM-155) were >128, 64, and > 128 microg/ml, respectively. Pyrosequence analysis determined the true identity of the beta-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.

Establishment of in vitro susceptibility testing methodologies and comparative activities of piperacillin in combination with the penem {beta}-lactamase inhibitor BLI-489.

The novel bicyclic penem inhibitor BLI-489 has demonstrated activity as an inhibitor of class A, C, and D beta-lactamases. To determine the combination of piperacillin and BLI-489 to be used in susceptibility testing that would most accurately identify susceptible and resistant isolates, a predictor panel of beta-lactamase-producing bacteria was utilized to determine the reliability of the combination of piperacillin-BLI-489 at a constant inhibitor concentration of 2 or 4 microg/ml and at ratios of 1:1, 2:1, 4:1, and 8:1. There were a number of strains that would be falsely reported as susceptible or intermediate if tested with the ratios of 1:1 and 2:1, whereas the constant concentration of 2 microg/ml of BLI-489 and the ratio of 8:1 had a tendency to overpredict resistance. Similar MICs were obtained with piperacillin-BLI-489 in a 4:1 ratio and when BLI-489 was held constant at 4 microg/ml. Based on these results, an in vitro testing methodology employing a constant concentration of 4 microg/ml BLI-489 was used to evaluate the combination of piperacillin-BLI-489 against a larger panel of recently identified clinical isolates. Approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MIC > or = 32 microg/ml). However, 92% of these piperacillin nonsusceptible strains were inhibited by < or =16 microg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by < or =16 microg/ml piperacillin-tazobactam. The combination of piperacillin-BLI-489 also demonstrated improved activity compared to that of piperacillin-tazobactam against the problematic extended-spectrum beta-lactamase- and AmpC-expressing strains.

Occurrence of tetracycline resistance genes among Escherichia coli isolates from the phase 3 clinical trials for tigecycline.

Tigecycline, a member of the glycylcycline class of antibiotics, was designed to maintain the antibacterial spectrum of the tetracyclines while overcoming the classic mechanisms of tetracycline resistance. The current study was designed to monitor the prevalence of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(M) resistance determinants in Escherichia coli isolates collected during the worldwide tigecycline phase 3 clinical trials. A subset of strains were also screened for the tet(G), tet(K), tet(L), and tet(Y) genes. Of the 1,680 E. coli clinical isolates screened for resistance to classical tetracyclines, 405 (24%) were minocycline resistant (MIC > or = 8 microg/ml) and 248 (15%) were tetracycline resistant (MIC > or = 8 microg/ml) but susceptible to minocycline (MIC < or = 4 microg/ml). A total of 452 tetracycline-resistant, nonduplicate isolates were positive by PCR for at least one of the six tetracycline resistance determinants examined. Over half of the isolates encoding a single determinant were positive for tet(A) (26%) or tet(B) (32%) with tet(C), tet(D), tet(E), and tet(M), collectively, found in 4% of isolates. Approximately 33% of the isolates were positive for more than one resistance determinant, with the tet(B) plus tet(E) combination the most highly represented, found in 11% of isolates. The susceptibilities of the tetracycline-resistant strains to tigecycline (MIC(90), 0.5 microg/ml), regardless of the encoded tet determinant(s), were comparable to the tigecycline susceptibility of tetracycline-susceptible strains (MIC(90), 0.5 microg/ml). The results provide a current (2002 to 2006) picture of the distribution of common tetracycline resistance determinants encoded in a globally sourced collection of clinical E. coli strains.

In vitro antibacterial activities of tigecycline and comparative agents by time-kill kinetic studies in fresh Mueller-Hinton broth.

Time-kill kinetics performed with tigecycline, in fresh MHB, demonstrated a consistent 1 to 2 log(10) CFU/ml reduction in bacterial counts against the majority of clinically relevant pathogens tested. Although classified as a bacteriostatic agent, tigecycline shows bactericidal activity against select isolates associated with serious infection. In general, vancomycin and imipenem demonstrated bactericidal activity.

Validation and reproducibility assessment of tigecycline MIC determinations by Etest.

A multicenter study was conducted to validate Etest tigecycline compared to the Clinical Laboratory Standards Institute reference broth microdilution and agar dilution methodologies. A large collection of gram-negative (n = 266) and gram-positive (n = 162) aerobic bacteria, a collection of anaerobes (n = 385), and selected collections of nonpneumococcal streptococci (n = 369), Streptococcus pneumoniae (n = 372), and Haemophilus influenzae (n = 372) were tested. Strains with reduced susceptibility to tigecycline were used with all test methods. The Etest showed excellent inter- and intralaboratory reproducibility for all organism groups tested regardless of the test methodology. The essential agreement values with the reference method (+/-1 dilution) were >99% for the collection of gram-negative and gram-positive aerobes; >98% for the S. pneumoniae, H. influenzae, and anaerobe collections; and 100% for the group of nonpneumococcal streptococci. These results validate the performance accuracy and utility of Etest tigecycline and verify the reproducibility of this convenient predefined gradient methodology for tigecycline susceptibility determination.

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli.

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.