Ceftazidime-resistant Strains Research Articles

Andrographolide stabilized-silver nanoparticles overcome ceftazidime-resistant Burkholderia pseudomallei: study of antimicrobial activity and mode of action

Burkholderia pseudomallei (B. pseudomallei) is a Gram-negative pathogen that causes melioidosis, a deadly but neglected tropical disease. B. pseudomallei is intrinsically resistant to a growing list of antibiotics, and alternative antimicrobial agents are being sought with urgency. In this study, we synthesize andrographolide-stabilized silver nanoparticles (andro-AgNPs, spherically shaped with 16 nm average diameter) that show excellent antimicrobial activity against B. pseudomallei, including ceftazidime-resistant strains, being 1–3 orders of magnitude more effective than ceftazidime and 1–2 orders of magnitude more effective than other green-synthesized AgNPs. The andro-AgNPs are meanwhile non-toxic to mammalian cell lines. The mode of action of Andro-AgNPs toward B. pseudomallei is unraveled by killing kinetics, membrane neutralization, silver ions (Ag+) release, reactive oxygen species (ROS) induction, membrane integrity, and cell morphology change studies. The antimicrobial activity and mode of action of andro-AgNPs against B. pseudomallei reported here may pave the way to alternative treatments for melioidosis.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy

IntroductionImmune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. MethodsThe imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. ResultsIn the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (−3.45 and −3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (−1.66 and −1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (−2.37log10CFU/g, P=0.1, or −1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. ConclusionsThese results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Pharmacodynamics of ceftazidime plus tobramycin combination dosage regimens against hypermutable Pseudomonas aeruginosa isolates at simulated epithelial lining fluid concentrations in a dynamic in vitro infection model

Hypermutable Pseudomonas aeruginosa strains are a major challenge in cystic fibrosis. We investigated bacterial killing and resistance emergence for approved ceftazidime and tobramycin regimens, alone and in combination. Pseudomonas aeruginosa PAOΔmutS and six hypermutable clinical isolates were examined using 48-h static concentration time-kill (SCTK) studies (inoculum ~107.5 CFU/mL); four strains were also studied in a dynamic in vitro model (IVM) (inoculum ~108 CFU/mL). The IVM simulated concentration-time profiles in epithelial lining fluid following intravenous administration of ceftazidime (3 g/day and 9 g/day continuous infusion), tobramycin (5 mg/kg and 10 mg/kg via 30-min infusion 24-hourly; half-life 3.5 h), and their combinations. Time courses of total and less-susceptible populations were determined. Ceftazidime plus tobramycin demonstrated synergistic killing in SCTK studies for all strains, although to a lesser extent for ceftazidime-resistant strains. In the IVM, ceftazidime and tobramycin monotherapies provided ≤5.4 and ≤3.4 log10 initial killing, respectively; however, re-growth with resistance occurred by 72 h. Against strains susceptible to one or both antibiotics, high-dose combination regimens provided >6 log10 initial killing, which was generally synergistic from 8-24 h, and marked suppression of re-growth and resistance at 72 h. The time course of bacterial density in the IVM was well described by mechanism-based models, enabling Monte Carlo simulations (MCSs) to predict likely effectiveness of the combination in patients. Results of the IVM and MCS suggested antibacterial effect depends both on the strain's susceptibility and hypermutability. Further investigation of the combination against hypermutable P. aeruginosa strains is warranted.

Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy

IntroductionImmune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. MethodsThe imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. ResultsIn the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (−3.45 and −3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (−1.66 and −1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (−2.37log10CFU/g, P=0.1, or −1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. ConclusionsThese results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Efficacy of ceftolozane/tazobactam, alone and in combination with colistin, against multidrug-resistant Pseudomonas aeruginosa in an in vitro biofilm pharmacodynamic model

ObjectivesCeftolozane/tazobactam is a potential tool for infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa), but its efficacy against some difficult-to-treat infections has not been well defined. MethodsUsing an in vitro pharmacodynamic biofilm model, this study evaluated the comparative efficacy of ceftolozane/tazobactam against MDR/extensively drug-resistant (XDR) P. aeruginosa strains, alone and in combination with colistin. Simulated regimens of ceftolozane/tazobactam (2 g/1 g every 8 h), meropenem (2 g every 8 h) and ceftazidime (2 g every 8 h), alone and in combination with colistin (continuous infusion) were evaluated against three colistin-susceptible and ceftazidime-resistant strains: MDR-HUB1, ceftolozane/tazobactam-susceptible and meropenem-susceptible; XDR-HUB2, ceftolozane/tazobactam-susceptible and meropenem-resistant; MDR-HUB3, ceftolozane/tazobactam-resistant and meropenem-susceptible. Antibiotic efficacy was evaluated by decreases in bacterial counts (Δlog CFU/mL) from biofilm-embedded bacteria over 54 h. Resistance emergence was screened. ResultsAmong monotherapies, ceftolozane/tazobactam had low killing but no resistance appeared, ceftazidime was ineffective, colistin was initially effective but regrowth and resistance occurred, and meropenem was bactericidal against carbapenem-susceptible strains. Ceftolozane/tazobactam plus colistin was the most effective combination against the meropenem-resistant XDR-HUB2 strain (Δlog CFU/mL 54–0 h = –4.42 vs. –3.54 for meropenem-colistin; P = 0.002), whereas this combination against MDR-HUB1 (–4.36) was less effective than meropenem-colistin (–6.25; P < 0.001). Ceftolozane/tazobactam plus colistin was ineffective against the ceftolozane/tazobactam-resistant strain; meropenem plus colistin was the most bactericidal therapy (–6.37; P < 0.001 vs. others). Combinations of active beta-lactams plus colistin prevented the emergence of colistin-resistant strains. ConclusionsCombinations of colistin plus ceftolozane/tazobactam and meropenem were the most appropriate treatments for biofilm-related infections caused by XDR and MDR P. aeruginosa strains, respectively. These combinations could be considered as potential treatment options for these difficult to treat infections.

Phenotypic and Molecular Detection of Extended-spectrum beta-lactamase among Ceftazidime Resistant Pseudomonas aeruginosa Isolated from Wound Swab.

ESBL producing Pseudomonas aeruginosa has been reported to be an important cause of nosocomial infection. The study was undertaken to determine ESBL producing Pseudomonas aeruginosa by phenotypic method and to detect bla OXA-10 ESBL gene by molecular method. A total of 288 wound infection cases attending the in-patient department of Mitford Hospital and Dhaka Medical College Hospital were enrolled for this study. Pseudomonas aeruginosa was isolated following standard procedure and subjected to antimicrobial susceptibility test by disc-diffusion method. Ceftazidime resistant strains were confirmed by MIC by agar dilution method. Confirmed ceftazidime resistant strains were tested for ESBL production by CLSI phenotypic confirmatory disc diffusion test (PCDDT) and bla OXA-10 ESBL gene was identified by employing conventional PCR. Out of 92 Pseudomonas aeruginosa, confirmed ceftazidime resistant strains were 60. PCDDT detected 49 (81.67%) ESBL producers and PCR detected 44 (73.33%) positive strains for bla OXA-10 ESBL gene among 60 ceftazidime resistant strains of Pseudomonas aeruginosa. The study showed high proportion of ESBL producers among ceftazidime resistant Pseudomonas aeruginosa.&#x0D; Bangladesh J Med Microbiol 2018; 12 (1): 15-19

Pharmacodynamics of ceftazidime/avibactam against extracellular and intracellular forms of Pseudomonas aeruginosa.

When tested in broth, avibactam reverses ceftazidime resistance in many Pseudomonas aeruginosa that express ESBLs. We examined whether similar reversal is observed against intracellular forms of P. aeruginosa . Strains: reference strains; two engineered strains with basal non-inducible expression of AmpC and their isogenic mutants with stably derepressed AmpC; and clinical isolates with complete, partial or no resistance to reversion with avibactam. Pharmacodynamic model: 24 h concentration-response to ceftazidime [0.01-200 mg/L alone or with avibactam (4 mg/L)] of bacteria in broth or bacteria phagocytosed by THP-1 monocytes, with calculation of ceftazidime relative potency ( C s : concentration yielding a static effect) and maximal relative effect [ E max : cfu decrease at infinitely large antibiotic concentrations (efficacy in the model)] using the Hill equation. Cellular content of avibactam: quantification by LC-MS/MS. For both extracellular and intracellular bacteria, ceftazidime C s was always close to its MIC. For ceftazidime-resistant strains, avibactam addition shifted ceftazidime C s to values close to the MIC of the combination in broth. E max was systematically below the detection limit (-5 log 10 ) for extracellular bacteria, but limited to -1.3 log 10 for intracellular bacteria (except for two isolates) with no effect of avibactam. The cellular concentration of avibactam reflected extracellular concentration and was not influenced by ceftazidime (0-160 mg/L). The potential for avibactam to inhibit β-lactamases does not differ for extracellular and intracellular forms of P. aeruginosa , denoting an unhindered access to its target in both situations. The loss of maximal relative efficacy of ceftazidime against intracellular P. aeruginosa was unrelated to resistance via avibactam-inhibitable β-lactamases.

Rapid Detection of Antibiotic Resistance in Gram-Negative Bacteria Through Assessment of Changes in Cellular Morphology.

Rapid antimicrobial susceptibility testing has the potential to improve patient outcomes and reduce healthcare-associated costs. In this study, a novel assay based on bacterial cell elongation after exposure to an antibiotic (ceftazidime) was evaluated for its ability to rapidly detect resistance in Gram-negative bacteria. The assay was used to detect resistance in a large collection of strains containing 320 clinical isolates of Acinetobacter baumannii, 171 clinical isolates of Klebsiella pneumoniae, and 212 clinical isolates of Pseudomonas aeruginosa, and the results were compared to those obtained using standard antimicrobial susceptibility testing methods. The assay identified ceftazidime-resistant strains with 100% sensitivity and 100% specificity for A. baumannii, 100% sensitivity and 97.2% specificity for K. pneumoniae, and with 82.3% sensitivity and 100% specificity for P. aeruginosa. Importantly, results were obtained in 1 hour 15 minutes from exponentially growing cultures. This study demonstrates that changes in cell length are highly correlated with phenotypic antibiotic susceptibility determined using standard susceptibility testing methods. This study therefore provides proof-of-concept that changes in cell morphology can be used as the basis for rapid detection of antibiotic resistance and provides the basis for the development of novel rapid diagnostics for the detection of antibiotic resistance.

Antimicrobial Peptide Novicidin Synergizes with Rifampin, Ceftriaxone, and Ceftazidime against Antibiotic-Resistant Enterobacteriaceae In Vitro

The spread of antibiotic resistance among Gram-negative bacteria is a serious clinical threat, and infections with these organisms are a leading cause of mortality worldwide. Traditional novel drug development inevitably leads to the emergence of new resistant strains, rendering the new drugs ineffective. Therefore, reviving the therapeutic potentials of existing antibiotics represents an attractive novel strategy. Novicidin, a novel cationic antimicrobial peptide, is effective against Gram-negative bacteria. Here, we investigated novicidin as a possible antibiotic enhancer. The actions of novicidin in combination with rifampin, ceftriaxone, or ceftazidime were investigated against 94 antibiotic-resistant clinical Gram-negative isolates and 7 strains expressing New Delhi metallo-β-lactamase-1. Using the checkerboard method, novicidin combined with rifampin showed synergy with >70% of the strains, reducing the MICs significantly. The combination of novicidin with ceftriaxone or ceftazidime was synergistic against 89.7% of the ceftriaxone-resistant strains and 94.1% of the ceftazidime-resistant strains. Synergistic interactions were confirmed using time-kill studies with multiple strains. Furthermore, novicidin increased the postantibiotic effect when combined with rifampin or ceftriaxone. Membrane depolarization assays revealed that novicidin alters the cytoplasmic membrane potential of Gram-negative bacteria. In vitro toxicology tests showed novicidin to have low hemolytic activity and no detrimental effect on cell cultures. We demonstrated that novicidin strongly rejuvenates the therapeutic potencies of ceftriaxone or ceftazidime against resistant Gram-negative bacteria in vitro. In addition, novicidin boosted the activity of rifampin. This strategy can have major clinical implications in our fight against antibiotic-resistant bacterial infections.

Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa.

The antipseudomonal efficiency and mechanism of action of a novel engineered antimicrobial peptide, T9W, were evaluated in this study. T9W displayed high activity, with a lethal concentration (LC) of 1 to 4 μM against Pseudomonas aeruginosa, including against ciprofloxacin-, gentamicin-, and ceftazidime-resistant strains, even in the presence of 50 to 300 mM NaCl, 1 to 5 mM Ca(2+), or 0.5 to 2 mM Mg(2+). The time-kill curve (TKC) analysis demonstrated concentration-dependent activity, with T9W achieving complete killing in less than 30 min at 1× LC and in less than 5 min at 4× LC. Combination TKC analyses additionally demonstrated a synergistic effect with ciprofloxacin and gentamicin. The selectivity of T9W was further supported by its ability to specifically eliminate P. aeruginosa in a coculture with macrophages without toxicity to the mammalian cells. The results from fluorescent measurement indicated that T9W bound to lipopolysaccharide (LPS) and induced P. aeruginosa membrane depolarization, and microscopic observations and flow cytometry further indicated that T9W targeted the P. aeruginosa cell membrane and disrupted cytoplasmic membrane integrity, thereby causing cellular content release leading to cell death. This study revealed the potential usefulness of T9W as a novel antimicrobial agent against P. aeruginosa.

Activity of ceftazidime-avibactam against fluoroquinolone-resistant Enterobacteriaceae and Pseudomonas aeruginosa.

Ceftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200 Enterobacteriaceae and 25 Pseudomonas aeruginosa strains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistant Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBL Escherichia coli (MIC90 of 0.25 mg/liter), ESBL Klebsiella pneumoniae (MIC90 of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90 of 1 mg/liter), non-ESBL E. coli (MIC90 of ≤0.125 mg/liter), non-ESBL K. pneumoniae (MIC90 of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90 of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistant P. aeruginosa strains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90 of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtained in vitro from two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90 values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains of Enterobacteriaceae and P. aeruginosa were ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affect Enterobacteriaceae and P. aeruginosa susceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

In vitro activity of ceftazidime-avibactam combination in in vitro checkerboard assays.

To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobacter cloacae) and 26 strains of Pseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n=69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC50 and MIC90 being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases.

Determination of vancomycin minimum inhibitory concentration for ceftazidime resistant Streptococcus pneumoniae in Iran.

BackgroundIn the context of growing health concerns over antibiotic resistance, the evaluation of the minimum inhibitory concentration (MIC) of vancomycin for Streptococcus pneumoniae (S. pneumoniae) strains resistant to ceftazidime becomes important for guiding health policy makers. The aim of this study was to determine vancomycin MIC of ceftazidime resistant S. pneumoniae strains.MethodsFifty identified serotypes of ceftazidime resistant S. pneumoniae strains were included in the study. The vancomycin MIC of the above mentioned bacteria was determined based on the 0.5 McFarland standards, by using a microdilution broth and the Etest method.ResultsThe results showed that out of 50 ceftazidime resistant strains of S. pneumoniae, 46 strains (92%) have shown a vancomycin MIC ≤0.19 − 0.1.5 μg/ml and only four strains (8%) have shown a vancomycin MIC equal to 1.5 μg/ml and the related maximum zone of inhibition was of 10 millimeter diameters.ConclusionsThe results of this investigation point out the emergence of S. pneumoniae strains with a vancomycin MIC ≥1.5 μg/ml, which were resistant to ceftazidime. This finding uncovers a major health concern: a vancomycin MIC higher than 1.5 μg/ml and maximum zone of inhibition of only 10 millimeter. These findings represent an important warning for health authorities globally, concerning the treatment of patients, as the occurrence of S. pneumoniae strains with decreased vancomycin susceptibility has been demonstrated.

Rapid emergence of a ceftazidime-resistant Burkholderia multivorans strain in a Cystic Fibrosis patient

Burkholderia multivorans poses a serious health threat to cystic fibrosis patients due to innate resistance to multiple antibiotics and acquisition of resistance to a range of antibiotics due to the frequent use of antibiotics to treat chronic infections. Monitoring antibiotic susceptibility is crucial to managing patient care. We identified the rapid emergence of a ceftazidime-resistant strain in a single patient within four days during a hospitalization for treatment of an exacerbation. B. multivorans was isolated from expectorated sputum samples using Burkholderia cepacia selective agar. A macrodilution assay was performed on all isolates to determine the minimum inhibitory concentration of ceftazidime. Approximately 4000 colonies were scored to identify the percent of ceftazidime-resistant colonies. Extracted DNA was used to determine the total bacterial counts and abundance of B. multivorans using quantitative PCR. An increase from no detectable B. multivorans ceftazidime-resistant colonies to over 75% of all colonies tested occurred within a four-day period. The resistant population remained dominant in 6 of the 8 samples in the following 17 months of the study. qPCR revealed an association between change in the percent of resistant colonies and abundance of B. multivorans, but not of total bacteria. No association was found between the acquisition of resistance to ceftazidime and other antibiotics commonly used to treat B. multivorans infections. The rapid emergence of a ceftazidime-resistant by B. multivorans strain occurred during a hospitalization while under selective pressure of antibiotics. The resistant strain maintained dominance in the B. multivorans population which resulted in an overall decline in a patient health and treatment efficacy.

Third generation cephalosporin-resistance in Klebsiella pneumoniae isolates: an emerging threat

Background: Newer generation cephalosporin-resistance among Klebsiella pneumoniae organisms has increased recently. Present study is undertaken to find incidence, antimicrobial susceptibility and prevalence of extended spectrum beta-lactamase (ESBL) in K. pneumoniae isolates in a tertiary care hospital. Methods: Prospective study was carried out between June to December 2011. Samples of pus, blood, urine, cerebro-spinal fluid, stool, peritoneal, pleural and synovial fluid were collected from indoor and outdoor patients for isolation and antimicrobial susceptibility pattern of K. pneumoniae in the department of microbiology, G.R. Medical College Gwalior, M.P. Ceftazidime resistant K. pneumoniae were subjected to Phenotypic Confirmatory Disc Diffusion Test (PCDDT) and Double Disc Synergy Test (DDST) for detection of ESBL. Results: Out of 2480 samples collected a total of 530 K. pneumoniae were isolated and subjected to antimicrobial susceptibility. Antibiotic sensitivity to imipenem, cefoperazone, amikacin and ofloxacin were 82, 74, 73 and 72% respectively whereas sensitivity to ceftizoxime, ceftriaxone cefotaxime, ceftazidime ranged between 47-50%. K. pneumoniae were found to be resistant to ampicillin, co-trimoxazole, doxycycline and gentamicin, by 91, 82, 54 and 50% respectively. Among third generation cephalosporins K. pneumoniae were least sensitive (47%) to ceftazidime. About 33 and 32% of the ceftazidime resistant strains were found to be ESBL positive by PCDDT and DDST respectively. Conclusions: This study has shown that prevalence of ESBL producing K. pneumoniae is the most important reason for increased resistance to third generation cephalosporins. There is need to carry out tests for detection of ESBL producing bacteria routinely.

Detección de la β-lactamasa de espectro extendido OXA-141 en cepas de Pseudomonas aeruginosa aisladas de pacientes con fibrosis quística

The aims of this research were to study the presence of extended spectrum β-lactamases (ESBL) to investigate the location of the genes encoding these enzymes, and determine the clonal relationship of strains of ceftazidime-resistant Pseudomonas aeruginosa isolated from Mexican patients with cystic fibrosis. We determined the resistance profile to 11 antibiotics (CLSI) and phenotypic ESBL detection following a disk diffusion method adapted for P. aeruginosa. Characterization of ESBL genes and integrons was performed by polymerase chain reaction (PCR) and DNA sequencing, while analysis of the clonal relationship was performed by pulsed field gel electrophoresis (PFGE). Of the 148 strains studied, 22 were resistant to ceftazidime, and analysis by PCR and sequencing revealed the presence of the gene bla(OXA-141) in 7 strains, 6 of which were resistant and one, susceptible to ceftazidime. In addition, bla(GES) was detected in 11 strains. intI2 and intI3 genes were not detected by PCR, but in the 6 ceftazidime-resistant strains, the bla(OXA-141) gene was determined in a class 1 integron. Analysis of the clonal relationship of isolates showed that the majority of patients were infected during the study period with P. aeruginosa strains that exhibit different patterns, especially in individuals without a familial relationship. This report demonstrates the existence of the bla(OXA-141) gene associated with a class 1 integron in several strains of P. aeruginosa, as well as bla(GES) genes, and their location and variants are being studied by our research group. This, combined with the diversity of strains able to infect several susceptible individuals, suggests the risk of spread of P. aeruginosa-strain ESBL producers among Mexican populations with cystic fibrosis.

Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov.

Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3 T, CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase β-subunit and RNA polymerase β-subunit, genomic DNA–DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3 T (CCUG 58127 T = CECT 7729 T) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta.

Strain-Tailored Double-Disk Synergy Test Detects Extended-Spectrum Oxacillinases in Pseudomonas aeruginosa

The prevalence of class D extended-spectrum oxacillinases (ES-OXAs) in ceftazidime-resistant strains of Pseudomonas aeruginosa is often underestimated by double-disk synergy tests (DDST) using clavulanate. A DDST with a customized distance between a disk of ceftazidime or cefepime and inhibitors (clavulanate and imipenem) detected 14 out of 15 different ES-OXAs.

Tigecycline activity tested against multidrug-resistant Enterobacteriaceae and Acinetobacter spp. isolated in US medical centers (2005–2009)

We evaluated the activity of tigecycline against Enterobacteriaceae (9563 isolates) and Acinetobacter spp. (835) with various resistance phenotypes collected from 31 US medical centers in 2005–2009. The isolates were tested for susceptibility by the reference broth microdilution method against tigecycline and various comparators. Among Escherichia coli and Klebsiella spp., 6.8% and 15.4% exhibited an extended-spectrum β-lactamase (ESBL) phenotype, respectively; and 22.2% of Enterobacter spp. strains were ceftazidime-resistant. Tigecycline was active against E. coli [minimum inhibitory concentration (MIC 50/90), 0.12/0.25 μg/mL; 100.0% susceptible] independent of ESBL phenotype or resistance to other antimicrobials. Among Klebsiella spp., 97.9% of ESBL-producing Klebsiella spp. and 98.2% of imipenem–non-susceptible strains were susceptible to tigecycline (MIC 50/90, 0.5/1 μg/mL for both subsets). Tigecycline was active against Enterobacter spp. (MIC 50/90, 0.25/1 μg/mL; 98.4% susceptible), including ceftazidime-resistant strains (MIC 50/90, 0.5/2 μg/mL; 97.1% susceptible). Tigecycline inhibited 94.4% of Acinetobacter spp. overall (MIC 50/90, 0.5/2 μg/mL) and 86.2% of imipenem–non-susceptible (MIC 50/90, 1/4 μg/mL) strains at ≤2 μg/mL. No trend toward decreased tigecycline activity overtime was observed for any of the organisms or resistant subsets during the study period. These results indicate that tigecycline has sustained potent in vitro activity and a broad spectrum against these clinically important Gram-negative pathogens causing infections in US medical centers, including multidrug-resistant organism subsets.