COLICINE K : VI. THE IMMUNE RESPONSE OF HORSES TO A COLICINOGENIC STRAIN OF ESCHERICHIA COLI

1. The immunization of horses with the colicinogenic bacillus E. coli K235 L + O(m) stimulates antibodies which precipitate and neutralize colicine K and neutralize the heterologous colicine I as well. 2. Unlike rabbits, horses evoke predpitating antibodies for the sialic acid-containing polysaccharide colominic acid.     

COLICINE K : IV. THE EFFECT OF METABOLITES UPON COLICINE SYNTHESIS

The synthesis of colicine K by the colicinogenic bacillus E. coli K₂₃₅ has been studied in the chemostat. It has been found that colicine production is influenced by the pH of the medium, by the nature of the primary carbon source, and by the generation time of the microorganism.     

Colicine K. II. The preparation and properties of a substance having colicine K activity

By chemical fractionation a substance having colicine K activity has been obtained from the culture medium of E. coli K₂₃₅ L₊O. Colicine K activity was found associated with the O antigen of this microorganism. When the O antigen was dissociated, colicine K activity remained with the protein component of the antigen.     

Colicine K. III. The immunological properties of a substance having colicine K activity

By immunological means it has been shown that colicine K is associated with the O antigen of the colicinogenic bacillus E. coli K₂₃₅ L₊OC₊. The colicine K-O antigen complex elicits the formation of at least two types of antibodies, one a precipitin, the other a colicine-neutralizing antibody. The first precipitates colicine K without neutralizing it, the second neutralizes the colicine without precipitating it. Unlike the purified colicine K complex, the colicine protein component of the O antigen is precipitable by the neutralizing antibody. There is no demonstrable serological relationship between colicine K and phage T₆. These two agents must be considered to be separate and distinct entities.     

Characterization of the New AmpC β-Lactamase FOX-8 Reveals a Single Mutation,Phe313Leu,Located in the R2 Loop That Affects Ceftazidime Hydrolysis

A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in k_cat/K_m for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.     

A study of the antigenicity of T3 and T4 coli-dysentery bacteriophages during the vegetative stage of development

The development of viral neutralizing antibodies in animals injected with T₃ or T₄ phage is considerably inhibited by the presence of bacterial antigens. A new procedure has been described to liberate phage from infected E. coli B bacteria by inducing lysis with penicillin. By immunological means it has been shown that T₄-infected cultures of E. coli B, in which phage development has been inhibited with proflavine, contain the viral neutralizing antigen after lysis. In contrast, it has not been possible to demonstrate by immunological means the appearance of viral neutralizing antigen in E. coli B infected with T₃ prior to the appearance of intracellular phage.     

Colicine K. I. The production of colicine K in media maintained at constant pH

An apparatus for maintaining the pH of an actively growing bacterial culture at constant level has been described. Using this apparatus it has been shown that the production of maximal amounts of colicine K from a strain of E. coli known as K235 is dependent upon an enriched nutrient medium which is maintained at pH 7.0.     

PROPERTIES OF A BACTERIOPHAGE DERIVED FROM ESCHERICHIA COLI K235

A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants.     

Enhanced antibacterial activity of acid treated MgO nanoparticles on Escherichia coli

Acid treatment is one of the effective methods that directly modifies surface physical and chemical properties of inorganic materials, which improves the materials'' application potential. In this work, the surface modified MgO nanoparticles (NPs) were prepared through a facile acid-treatment method at room temperature. Compared with the untreated sample, the surviving Escherichia coli (E. coli, ATCC 25922) colonies of the modified MgO NPs decreased from 120 to 54 (10² CFU mL⁻¹). The enhanced antibacterial activity may be due to the improvement of oxygen vacancies and absorbed oxygen (O_A) content (from 41.6% to 63.1%) as confirmed by electron spin resonance (ESR) and X-ray photoelectron spectroscopy (XPS). These findings revealed that the acid treatment method could directly modify the surface of MgO NPs to expose more oxygen vacancies, which would promote reactive oxygen species (ROS) generation. The membrane tube and single ROS scavenging results further indicated that the increased antibacterial ability originated from the synergetic effect of ROS damage (especially ˙O₂⁻) and direct contact between H-MgO NPs and E. coli.

Acid treatment of MgO NPs increased surface oxygen vacancies, leading to more adsorbed oxygen and enhanced antibacterial activity on E. coli.     

Studies on bacteriophage. II. Inhibition of lysis of Escherichia coli B by the somatic antigen of Phase II Shigella sonnei

By serological means it has been shown that E. coli B contains an antigen closely related to the protein-lipocarbohydrate complex of Phase II Sh. sonnei. Lysis of E. coli B by three of the T viruses, T₃, T₄, and T₇, can be inhibited by the Phase II dysentery antigen. It has been suggested that the receptor of E. coli B with which these viruses combine is this newly described antigenic component. Two variants of the virus T₃ have also been described, in stocks which have been treated with the Phase II antigen. One of these variants infects both Phase II Sh. sonnei and E. coli B, and the other infects only the latter microorganism; neither of the two variants is inhibited by concentrations of the Phase II antigen of 1 mg. per cc. The distinctive properties of the variants are not hereditary.     

COLICIN K : VIII. THE IMMUNOLOGICAL PROPERTIES OF MITOMYCIN-INDUCED COLICIN K

Partially purified colicin K (mCol K) has been obtained from cultures of Escherichia coli K235 induced with mitomycin C. Unlike colicin K (Col K) derived from noninduced cultures of E. coli K235, mitomycin-induced colicin K (mCol K) is not associated with the type O-specific antigen of the colicinogenic bacillus. mCol K elicits in rabbits specific antibodies which precipitate and neutralize the homologous bacteriocin. These colicin-specific antibodies are not precipitated by the colicin-O antigen complex derived from noninduced bacteria. Colicin-neutralizing antibodies can be separated by zone electrophoresis into fractions having different electrophoretic mobilities. The antibodies with lower mobility strongly precipitate the homologous antigen mCol K; those with higher mobility neutralize the bacteriocin and form soluble antigen-antibody complexes.     

Epidemiology and Risk Factors for Isolation of Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamase in a Large U.S. Medical Center

A case-case-control study was conducted to identify independent risk factors for recovery of Escherichia coli strains producing CTX-M-type extended-spectrum β-lactamases (CTX-M E. coli) within a large Southeastern Michigan medical center. Unique cases with isolation of ESBL-producing E. coli from February 2010 through July 2011 were analyzed by PCR for bla_CTX-M, bla_TEM, and bla_SHV genes. Patients with CTX-M E. coli were compared to patients with E. coli strains not producing CTX-M-type ESBLs (non-CTX-M E. coli) and uninfected controls. Of 575 patients with ESBL-producing E. coli, 491 (85.4%) isolates contained a CTX-M ESBL gene. A total of 319 (84.6%) patients with CTX-M E. coli (282 [74.8%] CTX-M-15 type) were compared to 58 (15.4%) non-CTX-M E. coli patients and to uninfected controls. Independent risk factors for CTX-M E. coli isolation compared to non-CTX-M E. coli included male gender, impaired consciousness, H2 blocker use, immunosuppression, and exposure to penicillins and/or trimethoprim-sulfamethoxazole. Compared to uninfected controls, independent risk factors for isolation of CTX-M E. coli included presence of a urinary catheter, previous urinary tract infection, exposure to oxyimino-cephalosporins, dependent functional status, non-home residence, and multiple comorbid conditions. Within 48 h of admission, community-acquired CTX-M E. coli (n = 51 [16%]) and non-CTX-M E coli (n = 11 [19%]) strains were isolated from patients with no recent health care contacts. CTX-M E. coli strains were more resistant to multiple antibiotics than non-CTX-M E. coli strains. CTX-M-encoding genes, especially bla_CTX-M-15 type, represented the most common ESBL determinants from ESBL-producing E. coli, the majority of which were present upon admission. Septic patients with risk factors for isolation of CTX-M E. coli should be empirically treated with appropriate agents. Regional infection control efforts and judicious antibiotic use are needed to control the spread of these organisms.     

Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture,Japan

This study was performed to characterize CTX-M type extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 μg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of bla_CTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred bla_CTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.     

ESCHERICHIA COLI ASSOCIATED WITH COLOSTRUM-FREE NEONATAL PIGS RAISED IN ISOLATION

Escherichia coli 08 was the most frequent coliform isolated from the blood and liver of morbid and dead neonatal, colostrum-free piglets raised under extremely sanitary conditions. This strain accounted for 67 per cent of the typable E. coli. The next most numerous strain occurred at a frequency of 6 per cent. Hence, E. coli 08 was considered the main coli enteropathogen in our experimental, isolated environment. In random samples of the feces of healthy and diarrhetic neonatal piglets, 24 per cent of the typable E. coli was type 08. When a directed effort was made to isolate E. coli 08 from the feces of neonatal piglets in a healthy, colostrum-free litter, this strain was isolated from 17 per cent of the total E. coli colonies examined. Thus, the enteropathogen E. coli 08 was ubiquitous in the feces of piglets in our environment, making up approximately 20 per cent of the fecal E. coli. 85 per cent of the bacteremia and death in which E. coli was isolated from blood or liver occurred in piglets fed diets void in bovine and porcine gamma globulin. Tube agglutination tests demonstrated that agglutinins to E. coli 08, and other serotypes as well, were present in bovine colostrum and to a lesser extent in porcine colostrum. These agglutinins were practically lacking in solutions of porcine and bovine gamma globulin. Feeding 10⁹ E. coli 08 bacteria to 2-week-old, colostrum-free, gamma globulin-free, 08 agglutinin-free piglets did not produce visible disease.     

COLICINE K : V. THE SOMATIC ANTIGEN OF A NON-COLICINOGENIC VARIANT OF E. COLI K235

The somatic antigen of the non-colicinogenic bacillus E. coli K235 L^-OC^- has been isolated, and its chemical and serological properties have been compared with those of colicine K. The antigen of the non-colicinogenic bacillus has a protein content significantly lower than that of the C⁺ antigen, a difference which might be related to the antibacterial activity of the latter. The lipocarbohydrate components of the two antigens are chemically very similar; both contain the same proportions of galactose, glucose, heptose, rhamnose, glucosamine, and mannosamine. When tested by agar diffusion, the two antigens are indistinguishable, as are their lipocarbohydrate components. Our studies indicate that the bactericidal activity of colicine K does not reside in its lipocarbohydrate but in its protein component.     

THE NATURE OF COLICIN K FROM PROTEUS MIRABILIS

The colicinogenic factor K has been transferred from E. coli K 235 to Proteus mirabilis. The DNA of the colicinogenic Proteus has been shown to contain a small amount of a satellite DNA which presumably harbors the Col K factor. In the presence of mitomycin C the colicinogenic Proteus secretes colicin K into the growth medium. The bacteriocin has been purified by chromatography and obtained as an immunologically homogeneous substance unconjugated with other antigens of the Proteus bacillus. Proteus colicin K is a protein of relatively low molecular weight. It contains all of the usual amino acids except cysteine and is free of lipids and polysaccharides. The bacteriocin can be separated by electrofocusing into two major components. The latter have the same biological properties but differ in their specific electrical charges.     

In Vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical Escherichia coli isolates obtained from a patient. The CMY-2-producing E. coli isolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli isolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ec and CMY-2-Ec were identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33-Ec and CMY-2-Ec, respectively). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli strain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). The k_cat/K_m or inhibitor complex inactivation (k_inact)/K_{i app} (μM⁻¹ s⁻¹) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.     

Impact of Third-Generation-Cephalosporin Administration in Hatcheries on Fecal Escherichia coli Antimicrobial Resistance in Broilers and Layers

We investigated the impact of the hatchery practice of administering third-generation cephalosporin (3GC) on the selection and persistence of 3GC-resistant Escherichia coli in poultry. We studied 15 3GC-treated (TB) and 15 non-3GC-treated (NTB) broiler flocks and 12 3GC-treated (TL) and 10 non-3GC-treated (NTL) future layer flocks. Fecal samples from each flock were sampled before arrival on the farm (day 0), on day 2, on day 7, and then twice more. E. coli isolates were isolated on MacConkey agar without antibiotics and screened for 3GC resistance, and any 3GC-resistant E. coli isolates were further analyzed. 3GC-resistant E. coli isolates were found in all 3GC-treated flocks on at least one sampling date. The percentages of 3GC-resistant E. coli isolates were significantly higher in TB (41.5%) than in NTB (19.5%) flocks and in TL (49.5%) than in NTL (24.5%) flocks. In the day 2 samples, more than 80% of the E. coli strains isolated were 3GC resistant. 3GC-resistant E. coli strains were still detected at the end of the follow-up period in 6 out of 27 3GC-treated and 5 out of 25 non-3GC-treated flocks. Many 3GC-resistant E. coli strains were resistant to tetracycline, and there were significant differences in the percentages of resistance to sulfamethoxazole-trimethoprim, streptomycin, or gentamicin between treated and nontreated flocks. bla_CTX-M and bla_CMY-2 were the most frequently detected genes. These results clearly demonstrated that 3GC-resistant strains are introduced early in flocks and that the use of 3GC in hatcheries promotes the selection of 3GC-resistant E. coli. Measures must be implemented to avoid the spread and selection of 3GC-resistant strains.     

Clavulanic Acid: a Beta-Lactamase-Inhibiting Beta-Lactam from Streptomyces clavuligerus

A novel β-lactamase inhibitor has been isolated from Streptomyces clavuligerus ATCC 27064 and given the name clavulanic acid. Conditions for the cultivation of the organism and detection and isolation of clavulanic acid are described. This compound resembles the nucleus of a penicillin but differs in having no acylamino side chain, having oxygen instead of sulfur, and containing a β-hydroxyethylidine substituent in the oxazolidine ring. Clavulanic acid is a potent inhibitor of many β-lactamases, including those found in Escherichia coli (plasmid mediated), Klebsiella aerogenes, Proteus mirabilis, and Staphylococcus aureus, the inhibition being of a progressive type. The cephalosporinase type of β-lactamase found in Pseudomonas aeruginosa and Enterobacter cloacae P99 and the chromosomally mediated β-lactamase of E. coli are less well inhibited. The minimum inhibitory concentrations of ampicillin and cephaloridine against β-lactamase-producing, penicillin-resistant strains of S. aureus, K. aerogenes, P. mirabilis, and E. coli have been shown to be considerably reduced by the addition of low concentrations of clavulanic acid.     

IMP-51, a Novel IMP-Type Metallo-β-Lactamase with Increased Doripenem- and Meropenem-Hydrolyzing Activities,in a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate

A meropenem-resistant Pseudomonas aeruginosa isolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-β-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly). Escherichia coli expressing bla_IMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing bla_IMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.