Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.

Antibiotic-resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer1. Because new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics3-6 in which only a subset of cells are phenotypically resistant7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics ('multiple heteroresistance'). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.

Analogues of Disulfides from Allium stipitatum Demonstrate Potent Anti-tubercular Activities through Drug Efflux Pump and Biofilm Inhibition.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Antimicrobial Activity of Ceftazidime-Avibactam against Gram-Negative Bacteria Isolated from Patients Hospitalized with Pneumonia in U.S. Medical Centers, 2011 to 2015.

Bacterial isolates were collected from patients hospitalized with pneumonia (PHP), including ventilator-associated pneumonia (VAP), from 76 U.S. medical centers in 2011 to 2015. The Gram-negative organisms (n = 11,185, including 1,097 from VAP) were tested for susceptibility to ceftazidime-avibactam and comparators by the broth microdilution method. ß-Lactamase-encoding genes were screened using a microarray-based assay on selected isolates. Pseudomonas aeruginosa and Klebsiella spp. were the most common Gram-negative bacteria isolated from PHP and VAP. Ceftazidime-avibactam was very active against P. aeruginosa (n = 3,402; MIC50/MIC90, 2 and 4 µg/ml; 96.6% susceptible), including isolates nonsusceptible to meropenem (86.3% susceptible to ceftazidime-avibactam), piperacillin-tazobactam (85.6% susceptible), or ceftazidime (80.6% susceptible). Ceftazidime-avibactam was also highly active against Enterobacteriaceae (MIC50/MIC90, 0.12 and 0.5 µg/ml; 99.9% susceptible), including carbapenem-resistant Enterobacteriaceae (CRE) (n = 189; MIC50/MIC90, 0.5 and 2 µg/ml; 98.0% susceptible) and multidrug-resistant (MDR) (n = 674; MIC50/MIC90, 0.25 and 1 µg/ml; 98.8% susceptible) and extensively drug-resistant (XDR) (n = 156; MIC50/MIC90, 0.5 and 2 µg/ml; 98.1% susceptible) Enterobacteriaceae isolates, as well as Klebsiella species isolates showing an extended-spectrum ß-lactamase (ESBL) screening-positive phenotype (n = 433; MIC50/MIC90, 0.25 and 1 µg/ml; 99.5% susceptible). Among Enterobacter spp. (24.8% ceftazidime nonsusceptible), 99.8% of the isolates, including 99.4% of ceftazidime-nonsusceptible isolates, were susceptible to ceftazidime-avibactam. The most common ß-lactamases detected among Klebsiella pneumoniae and E. coli isolates were K. pneumoniae carbapenemase (KPC)-like and CTX-M-15, respectively. Only 8 of 6,209 Enterobacteriaceae isolates (0.1%) were ceftazidime-avibactam nonsusceptible, three NDM-1-producing strains with ceftazidime-avibactam MIC values of >32 µg/ml and five isolates with ceftazidime-avibactam MIC values of 16 µg/ml and negative results for all ß-lactamases tested. Susceptibility rates among isolates from VAP were generally similar or slightly higher than those from all PHP.

Regional resistance surveillance program results for 12 Asia-Pacific nations (2011).

The Regional Resistance Surveillance program monitored susceptibility rates and developing resistance by geographic region, including 12 Asia-Pacific (APAC) countries. Reference broth microdilution methods for susceptibility/interpretations were applied, processing 5,053 strains. Among Staphylococcus aureus isolates (37% methicillin-resistant S. aureus [MRSA], highest in South Korea [73%]), linezolid (LZD), tigecycline (TIG), and vancomycin were 100% active, but 33 and 34% of strains were levofloxacin (LEV) or macrolide resistant, respectively. Streptococcus pneumoniae was most resistant to ß-lactams and macrolides (45%) but was LZD, LEV, and TIG susceptible (>98%). Extended-spectrum ß-lactamase (ESBL) phenotype rates in Escherichia coli and Klebsiella spp. were 48 and 47%, respectively, and were highest in Taiwan, at 75 to 91%. The best anti-ESBL-phenotype agents were amikacin (81 to 96% susceptible), colistin (COL; >98%), TIG (>98%), and carbapenems (81 to 97%). Pseudomonas aeruginosa showed ≥20% resistance to all drugs except COL (99% susceptible). In conclusion, endemic evolving antimicrobial resistances in APAC nations show compromised roles for many commonly used antimicrobials.

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster).

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera. The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA. The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines. In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.

Investigating the antimicrobial activity of natural honey and its effects on the pathogenic bacterial infections of surgical wounds and conjunctiva.

Antimicrobial activities of 10-100% (wt/vol) concentrations of new honey, stored honey, heated honey, ultraviolet-exposed honey, and heated stored honey were tested against common human pathogens, including Escherichia coli, Entrobacter cloacae, Pseudomonas aeruginosa, Shigella dysenteriae, Klebsiella sp., Haemophilus influenzae, Proteus sp., Staphylococcus aureus, Streptococcus hemolyticus group B, and Candida albicans. Antimicrobial activity of honey was tested in acidic, neutral, or alkaline media. These were compared with similar concentrations of glucose in nutrient broth. Surgical wounds were made on the dorsum of mice and infected with S. aureus or Klebsiella sp. The wounds were treated with local application of honey four times a day or appropriate antibiotics and compared with control values. Bacterial conjunctivitis due to E. coli, Proteus sp., S. aureus, Klebsiella sp., and P. aeruginosa was induced in rats. Conjunctival application of honey four times a day or appropriate antibiotics was used for treatment and compared with control values. Growth of all the isolates was completely inhibited by 30-100% honey concentrations. The most sensitive microbes were E. coli, P. aeruginosa, and H. influenzae. Glucose showed less antimicrobial activity than honey, and many microbes showed positive culture even in 100% glucose. Heating to 80 degrees C for 1 hour decreased antimicrobial activity of both new and stored honey. Storage of honey for 5 years decreased its antimicrobial activity, while ultraviolet light exposure increased its activity against some of the microorganisms. Antimicrobial activity of honey was stronger in acidic media than in neutral or alkaline media. Single doses of honey used to prepare the 60% concentration in nutrient broth were bacteriocidal for P. aeruginosa and bacteriostatic for S. aureus and Klebsiella sp. during certain periods. Local application of raw honey on infected wounds reduced redness, swelling, time for complete resolution of lesion, and time for eradication of bacterial infection due to S. aureus or Klebsiella sp. Its potency was comparable to that of local antibiotics. Honey application into infective conjunctivitis reduced redness, swelling, pus discharge, and time for eradication of bacterial infections due to all the isolates tested.

Influence of substrate composition and flow rate on growth of Azospirillum brasilense Cd in a co-culture with 3 sorghum rhizobacteria.

The ability of Azospirillum brasilense Cd to colonize the niche occupied by 3 bacterial strains previously isolated from sorghum rhizosphere was studied by means of the Biolog system. The isolates were identified by different methods as strains belonging to Pseudomonas putida, Stenotrophomonas maltophilia, and Klebsiella terrigena species. Several C sources, also chosen among the constituents of sorghum root exudates, were used to evaluate the metabolic profiles of Azospirillum and the sorghum rhizobacteria. Azospirillum brasilense Cd exploited the same class of C compounds as the sorghum rhizobacteria and overlapped in their niche requirements. Since structure and functioning of a microbial community are largely affected by the flow rate of nutrient supply, the competitive behavior of A. brasilense Cd was studied in a chemostat mixed culture under C-limited conditions using disodium succinate as C source. Only at high growth rates, i.e., when the C source was highly supplied, A. brasilense Cd appeared to be a good competitor and it became the dominant species, whereas at low growth rates, it was outnumbered by the other species. However, the coexistence of all the strains was always maintained, thus suggesting that interactions other than competition or a potential cross-feeding might occur within the mixed culture.

Study of growth requirements other than cysteine of naturally occurring Escherichia coli and Klebsiella spp. auxotrophic for cysteine.

Cysteine remains the preferred supplement for cultivation of Cys- auxotrophs in vitro. Methionine, which reduced cysteine requirements, and branched-chain amino acids, which decreased cysteine toxicity, were identified as the components of casein hydrolysate responsible for growth enhancement by this additive. Glutathione and DL-homocysteine can be substituted for cysteine. Accumulation of these compounds in patients with renal impairment may favor selection of Cys- strains in vivo.

Assessment of conventional and commercial methods for identification of clinical isolates of cysteine-requiring strains of Escherichia coli and Klebsiella species.

Cysteine-requiring strains of the family Enterobacteriaceae that are auxotrophic for this amino acid because of defects in the sulfur assimilatory pathway account for about 1.5% of urinary tract isolates of Escherichia coli and Klebsiella species. Forty Escherichia and eight Klebsiella cysteine-requiring strains were used to test the ease with which various test systems identified clinical isolates of cysteine auxotrophs. In a preliminary experiment, the growth yield of 10 cysteine-requiring E. coli in 10 solutions of commercially available peptones was in each instance less than that of prototrophic control and showed that these sources of nutrients were suboptimal for these strains. A significant proportion of the cysteine-requiring strains were not adequately identified by growth-dependent tests which used various peptones as a nutrient source. Problems were encountered with all test systems examined, which were as follows: conventional methods; the API 20E, Microbact, and Vitek systems; and two rapid methods for the identification of E. coli, the Rapidec coli and the beta-D-glucuronidase tests. The performance of the test systems was only partly improved when inocula were derived from appropriately supplemented media. However, the problems of the growth-dependent tests were resolved when a cysteine-supplemented suspension was used to inoculate each test system.

Significance of low-temperature growth associated with the fecal coliform response, indole production, and pectin liquefaction in Klebsiella.

In the genus Klebsiella, the growth respnse in nutient broth at 10 degrees C correlates inversely with the operational definition of a fecal coliform and not merely with the ability to grow at 44.5 degrees C. Of the fecal coliform-positive Klebsiella, 97% did not grow at 10 degrees C after 72 h of incubation. Conversely, 97% of the fecal coliform-negative isolates grew at 10 degrees C. The amount of growth at 10 degrees C varied among the fecal coliform-negative isolates and was found to correlate with indole production and pectin liquefaction. Low-temperature growth associated with specific biochemical tests can be used to differentiate several groups in the genus Klebsiella. Three main groups were discerned. Group I consists of indole-negative, pectin-nonliquefying, fecal coliform-positive isolates that do not grow at 10 degrees C. Group II isolates are differentiated from group I by a fecal-coliform-negative response and growth at 10 degrees C. Group III are indole-positive, pectin-liquefying, fecal coliform-negative isolates that grow at 10 degrees C. In our culture collection, isolates of group I are most frequently of human/animal clinical origins, whereas isolates of groups II and III are predominantly derived from the environment.