The threat of persistent bacteria and fungi contamination in tuberculosis sputum cultures.

BACKGROUND: Tuberculosis (TB) sputum culture contaminants make it difficult to obtain pure TB isolates.We aimed to study and identify persistent TB sputum culture contaminants post the standard laboratory pre-culture sample decontamination techniques. METHODS: This was a longitudinal study of TB sputum culture contamination for a cohort of TB patients on standard treatment at: baseline, during TB treatment and post TB treatment. Sputum samples were decontaminated with 1.5%NaOH and neutralized using 6.8 Phosphate buffer solution.Sputum was then inoculated into MGIT (mycobactrial growth indicator tube) supplemented with 0.8ml PANTA. A drop of each positive MGIT culture was sub cultured onto blood agar and incubated for 48 hours at 35 -37OC.Any growth was identified using growth characteristics and colony morphology. RESULTS: From October 2017 through May 2019;we collected 8645 sputum samples of which 8624(99.8%) were eligible and inoculated into MGIT where 2444(28.3%)samples were TB culture positive and 255(10.4%)were positive for contaminants: 237 none-tuberculosis bacteria, 12 fungi and 6 mixed(none-tuberculous bacteria+fungi). There was no statistically significant difference between none tuberculosis bacteria and fungi in the treatment (OR=1.4,95%CI:0.26-7.47,p=0.690) and the post treatment TB phases(OR=2.02,95%CI:0.38-10.79,p=0.411)Vs baseline. CONCLUSION: None-tuberculous bacteria and fungi dominate the plethora of TB sputum culture contamination and persist beyond the standard laboratory pre-culture decontamination algorithm.

Enhanced antibacterial effect of azlocillin in conjugation with silver nanoparticles against Pseudomonas aeruginosa.

In recent years, the problems associated with bacterial resistance to antibiotics caused nanodrugs to be considered as a new way for infectious diseases treatment. The main purpose of this study was to develop a new agent against Pseudomonas aeruginosa, a very difficult bacterium to treat, based on azlocillin antibiotic and silver nanoparticles (AgNPs). Azlocillin was conjugated with AgNPs by chemical methods and its antimicrobial activity was studied against P. aeruginosa using well diffusion agar method. Then, minimum inhibitory concentration and minimum bactericidal concentration of the new conjugate was specified with macro-dilution method. The animal study showed the considerable enhanced antibacterial effect of azlocillin in conjugation with AgNPs against P. aeruginosa in comparison with azlocillin alone, AgNPs alone and azlocillin in combination with AgNPs.

Reduction of contamination of mycobacterial growth indicator tubes using increased PANTA concentration.

We assessed the effect of a double concentration of supplemental polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) added to the Mycobacterial Growth Indicator Tube (MGIT) on contamination and positivity rates in 216 sputum cultures. Contamination rates were respectively 12.9% and 5.5% for samples processed using standard and double PANTA concentrations (P = 0.0001, McNemar's test). Thirty-five per cent of cultures performed using standard PANTA and 36.5% of those performed using two-fold PANTA concentrations were positive for Mycobacterium tuberculosis, compared to 25.9% of cultures inoculated on Ogawa medium. These results suggest that the use of MGIT with 2× PANTA may be useful in reducing culture contamination without reducing the diagnostic yield.

[Meningoencephalitis caused by Pseudomonas cepacia]. / Meningoencefalitis por Pseudomonas cepacia.

A case of meningoencephalitis of bacterial etiology caused by Pseudomonas cepacia was described. The strain was received at the Reference Laboratory of Bacterial Acute Respiratory Infections of "Pedro Kouri" Institute of Tropical Medicine, where its microbiological identification was confirmed. This isolation was a finding in an adult immunocompetent patient. The evolution was favourable with no sequelae for his future life. Pseudomona cepacia has been associated with respiratory infections in patients with cystic fibrosis. Patients with Pseudomonas cepacia may be asymptomatic or present fatal acute and fulminant infection.

Structure-activity relationships of different beta-lactam antibiotics against a soluble form of Enterococcus faecium PBP5, a type II bacterial transpeptidase.

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. beta-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel beta-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different beta-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Delta1-36 rPBP5). The soluble protein, Delta1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH(2)-terminal protein sequence was verified by amino acid sequencing. Using beta-lactams with different R1 side chains, we show that azlocillin has greater affinity for Delta1-36 rPBP5 than piperacillin and ampicillin (apparent K(i) = 7 +/- 0.3 microM, compared to 36 +/- 3 and 51 +/- 10 microM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4 M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K(i) = 51 +/- 15 microM) but is slower at acylating (apparent k(2) = 0.14 +/- 0.02 M(-1) s(-1)). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a beta-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.

[Bacterial meningitis and Pseudomonas aeruginosa: apropos of a case]. / Meningitis bacteriana y Pseudomonas aeruginosa: a propósito de un caso.

Meningitis caused by gram-negative bacilli increased since the 1970, with a higher incidence in small children. Within this group of infections, the meningitis caused by Pseudomonas sp is rare. The case of a 54-year-old patient with a clinical picture of meningitis is reported. Pseudomonas aeruginosa was isolated from the cerebrospinal fluid. The meningitis caused by Pseudomonas aeruginosa should be taken into consideration because of the severity of the clinical picture and the high mortality and increasing strain resistance.

(-)-Epigallocatechin-3-gallate induces contraction of the rat aorta by a calcium influx-dependent mechanism.

Although the consumption of tea has been associated with beneficial cardiovascular effects, (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin in this beverage has shown seemingly contradictory actions on vascular tissues, for example vasorelaxant activity that could contribute favourably to prevention of cardiovascular disease, and contractile activity that could act in the opposite direction. The purpose of the present work was to study the contractile effects of EGCG on isolated rat thoracic aorta rings and its effects on the cytosolic free [Ca(2+)] ([Ca(2+)](i)) measured with fura-2 in cultured rat aortic smooth muscle cell line. In partially depolarised (15 mM KCl) aortic rings EGCG (30-300 microM), (+/-)-BAY K 8644 (0.1 microM) and thapsigargin (1 microM) induced a Ca(2+)-dependent, endothelium-independent contraction associated with [Ca(2+)](i) elevation in RASMC. EGCG enhanced the responses elicited by (+/-)-BAY K 8644 and thapsigargin both in aortic rings and in RASMC. Nifedipine totally inhibited the (+/-)-BAY K 8644-induced contraction, but only partially blocked the contractile responses to EGCG and thapsigargin, while SKF 96365 abolished both responses. The effects of these channel blockers were associated with a decrease in [Ca(2+)](i) in RASMC. Re-introduction of Ca(2+) in the medium after depletion of intracellular Ca(2+) stores with thapsigargin in a Ca(2+)-free solution elicited a contraction of aortic rings and an increase in [Ca(2+)](i) in RASMC. In both cases, this response was partially sensitive to nifedipine, abolished by SKF 96365 and clearly enhanced by EGCG. These results suggest that EGCG induces a transient endothelium-independent contraction in the rat aorta, probably by increasing smooth vascular cell membrane permeability to Ca(2+) through both non-specific and dihydropyridine-sensitive Ca(2+) channels.

Characterization of the susceptibility of mycobacteria in BACTEC 12B media containing PANTA that had been supplemented with ceftazidime, and characterization of the individual components of PANTA in the presence of C18-carboxypropylbetaine.

C(18)-carboxypropylbetaine (CB-18) specimen processing has enhanced the diagnosis of mycobacterioses by smear, culture, and nucleic acid amplification. However, toxic side effects of CB-18 in liquid culture, especially in the presence of antibiotics, have been reported. The interaction of CB-18 at 20-25 microg/ml with the individual components of the antibiotic supplement PANTA that had been fortified with ceftazidime (PANTA-caz) was characterized in BACTEC 12B cultures using four mycobacterial isolates. When the Mycobacterium tuberculosis isolate ATCC 27294 was examined CB-18 plus PANTA-caz did not significantly alter the time-to-positive (i.e., time to a growth index (GI) of 15 (GI(15))), but did significantly increase the time to a GI of 500 (GI(500)) by approximately 8.5 days. This result could be attributed primarily to nalidixic acid, but also to ceftazidime to a lesser degree. Statistically significant increases in GI(15) of 12.5 days and GI(500) of 16.5 days were observed in the presence of CB-18 plus PANTA-caz with the Mycobacterium avium isolate ATCC 25291. These increases were due exclusively to trimethoprim. Statistically significant increases of approximately 2.5 and 9 days in GI(15) and GI(500), respectively, were observed with Mycobacterium kansasii ATCC 12478 in CB-18 plus PANTA-caz. The presence of nalidixic acid and ceftazidime were responsible for these alterations. When the behavior of the Mycobacterium fortuitum isolate ATCC 6841 was investigated in CB-18 plus PANTA-caz, significant increases in GI(15) of 8.5 days and GI(500) of 13 days were observed. The additive effects of nalidixic acid and azlocillin were responsible for these results. No single component of the PANTA-caz formulation was responsible for the interaction between CB-18 and PANTA-caz, although nalidixic acid contributed to these effects most often. These findings are consistent with the previous recommendation that CB-18 specimen processing follow a dilution-based format to ensure that the concentration of CB-18 carried-over into liquid media falls below 5-10 microg/ml.

[Azlocillin plus amikacin: an alternative therapy for sepsis caused by resistant staphylococci?]. / Azlocilina y amikacina: Una alternativa terapéutica para la septicemia por estafilococos resistentes?

OBJECTIVE: To assess the effectiveness of combined therapy with azlocillin and amikacin in a group of neonates with sepsis caused by multiresistant staphylococci who were hospitalized in the neonatal intensive care unit of Hospital Ginecobstétrico "America Arias" in Havana, Cuba, from 1998 to 2000. METHODS: A retrospective study was carried out of the clinical and laboratory results obtained in 15 patients with sepsis caused by multiresistant staphylococci who received combined therapy with azlocillin and amikacin, according to hospital guidelines on the use of antibiotics. We used a broth microdilution method to study the patterns of resistance shown by isolated strains to 10 of the antibiotics in use. In vitro synergy tests, specifically the checkerboard technique with microtitration plates, were used to observe the effects of treatment in 8 patients. RESULTS: Twelve coagulase-negative staphylococci and three Staphylococcus aureus isolates showed five different patterns of resistance on the basis of their sensitivity to oxacillin, three aminoglycosides, and vancomycin. Six of the synergy tests showed a considerable synergistic effect, with an average three-fold reduction in the minimum inhibitory concentrations (MIC) of the two antibiotics used to treat the patients. No antagonistic effects were noted, and the combined antibiotics showed an overall clinical effectiveness of 91.7%. CONCLUSIONS: The test showed that the therapeutic combination used was effective, but further studies are needed before conclusive results are obtained.

Reduction of contamination of mycobacterial growth indicator tubes with a modified antimicrobial combination.

Culture in the fluorimetric Mycobacteria Growth Indicator Tube (MGIT) treated with a combination of vancomycin, amphotericin B, and nalidixic acid (VAN) showed growth of most strains of 31 mycobacterial species with a less-than-1-day delay. The results were similar to those in the MGIT with polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin, but with respiratory specimens, the MGIT with VAN showed a lower contamination rate with no change in the detection rate or time.

Effect of beta-estradiol on voltage-gated Ca(2+) channels in rat hippocampal neurons: a comparison with dehydroepiandrosterone.

We investigated the effects of beta-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate on intracellular calcium concentration ([Ca(2+)](i)) increases induced by gamma-aminobutyric acid (GABA), high K(+) and N-methyl-D-aspartate acid (NMDA) in cultured hippocampal neurons. Acute treatment with beta-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate inhibited the GABA-induced [Ca(2+)](i) increases to the similar extent. Tamoxifen, an estrogen receptor antagonist, did not block the inhibitory effects of beta-estradiol. On the other hand, GABA type A (GABA(A)) receptor antagonists, picrotoxin and bicuculline, blocked the GABA-induced [Ca(2+)](i) increases. Previously, we demonstrated that GABA- and high K(+)-induced [Ca(2+)](i) increases were commonly mediated by voltage-gated calcium channels (VGCCs). Therefore, we examined the effects of these steroids on the high K(+)-induced [Ca(2+)](i) increases. The inhibitory effect of beta-estradiol on the high K(+)-induced [Ca(2+)](i) increases was much greater than that of dehydroepiandrosterone and dehydroepiandrosterone sulfate. beta-Estradiol inhibited the NMDA-induced [Ca(2+)](i) increases with an IC(50) of 51.8 microM and NMDA responses were reduced to half in the presence of 10 micro M nifedipine, indicating that the NMDA-induced [Ca(2+)](i) increases also involved VGCCs. Further, we examined the inhibitory effect of beta-estradiol on the high K(+)-induced [Ca(2+)](i) increases in the presence of a N-type VGCCs antagonist, 1 microM omega-conotoxin, or a L-type VGCCs antagonist, 10 microM nifedipine. The IC(50) value of beta-estradiol alone (45.5 microM) was similar to that of omega-conotoxin (33.1 microM), while the value combined with nifedipine was reduced to 2.2 microM. beta-Estradiol also abolished the positive modulatory effect of L-type VGCCs agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (Bay K 8644). Our results showed that the inhibitory mechanism of beta-estradiol is different from that of dehydroepiandrosterone and dehydroepiandrosterone sulfate and beta-estradiol may act primarily at L-type VGCCs.

An outbreak of multidrug-resistant Pseudomonas aeruginosa infection associated with contamination of bronchoscopes and an endoscope washer-disinfector.

Over a two-month period, two distinct types of Pseudomonas aeruginosa resistant to ceftazidime and azlocillin were isolated from bronchial specimens of ITU patients who had been previously bronchoscoped. The source of the outbreak was probably a faulty contaminated bronchoscope washer-disinfector which had been purchased a year earlier but not properly maintained. This paper describes the outbreak, the identification and elimination of the source, and the steps taken to prevent recurrence. Several automated, closed washer-disinfectors had been bought by the hospital in response to health and safety concerns about glutaraldehyde disinfection toxicity, but the operation and maintenance of these machines had not been supervised. Several other washer-disinfectors were also found to be faulty. The potential hazards of automated endoscope washer-disinfectors and the importance of controlled professional maintenance, servicing and training is discussed.

Ability of azlocillin and tobramycin in combination to delay or prevent resistance development in Pseudomonas aeruginosa.

The ability of combinations of azlocillin and tobramycin to prevent or delay resistance development in eight Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients was studied using chequerboard titration and in-vitro serial subculture. No isolate had developed resistance to tobramycin after 12 treatments with the antibiotic combination. Azlocillin resistance had not developed in four isolates after 16 exposures, and was delayed in the other four isolates for at least eight exposures. Beta-lactamase production was responsible for azlocillin resistance in two isolates and occurred to a lesser extent in a third.

Development of resistance and cross-resistance in Pseudomonas aeruginosa exposed to subinhibitory antibiotic concentrations.

The purpose of this study was to compare resistance and cross-resistance development in Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients to commonly used antipseudomonal antibiotics. Isolates were repeatedly exposed to subinhibitory concentrations of either azlocillin, tobramycin, ceftazidime or ciprofloxacin. On 10 consecutive occasions, samples were removed from the half-MIC well of a microtitre plate and regrown in drug-free medium to provide the next inoculum for MIC determination. The increase in MIC at the end of the treatment period was significant (p<0.05) for all selecting antibiotics. Cross-resistance to unrelated antibiotics was not observed, but was significant (p<0.05) in all beta-lactams (ticarcillin, piperacillin, ceftazidime and cefsulodin) studied where azlocillin was the selecting antibiotic. The addition of clavulanic acid to ticarcillin and of tazobactam to piperacillin had no effect on cross-resistance. The development of resistance to azlocillin was associated with increased beta-lactamase activity and a change in isoelectric point of the beta-lactamases. The result of this study supports a rotational policy for antipseudomonal antibiotics in CF patients.

FtsI and FtsW are localized to the septum in Escherichia coli.

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

Effects of decontamination methods and culture conditions on viability of Mycobacterium ulcerans in the BACTEC system.

We used the BACTEC system to evaluate the effects of several decontamination methods and the addition of antibiotics on the viability of Mycobacterium ulcerans. The effects of polyoxyethylene stearate or egg yolk as supplements were also evaluated to determine their impact on the growth of M. ulcerans. Strains of different geographic origins were subjected to Petroff, reversed Petroff, oxalic acid, and mild HCI treatments. After treatment, the viability of each strain was assessed in the BACTEC system. All of the decontamination methods tested adversely affected bacterial viability. Treatment with mild HCl gave the best results, allowing better growth rates with some strains and causing a delay in growth with others, depending on the geographic origin of the strain. A mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin did not significantly inhibit growth. Supplementing BACTEC medium with egg yolk markedly improved the recovery of M. ulcerans following the use of each of the decontamination methods. Our findings demonstrate a detrimental impact on the viability of M. ulcerans by all of the decontamination methods currently in common use. This explains, at least in part, the difficulty often experienced in cultivating this organism from clinical specimens. Egg yolk should be added to enhance the rate of successful primary cultivation of M. ulcerans in the BACTEC system.

[Isolation and biological characterization of Legionella pneumophila mutants resistant to aminoglycoside antibotics and their protective action]. / Poluchenie, biologicheskaya kharakteristika mutantov Legionella pneumophila k nekotorym aminoglikozidnym antibiotikam i ikh protektivnye svolstva.

Preliminary estimation of virulence in some antibiotic resistant mutants of Legionella pneumophila, Philadelphia 1 in various models of infection revealed its decrease in the mutants resistant to azlocillin, cefotaxime, fluoroquinolone LIB-80, neamine and streptomycin. Detailed investigation of the neamine resistant mutants showed that in relation to streptomycin susceptibility such mutants could be divided into 3 classes: susceptible to streptomycin, resistant to high concentrations of streptomycin and resistant to moderate concentrations of streptomycin. Part of mutants Nea(r)Strr and Nea(r)Strr500 and all mutants Nea(r)Strr100 proved to be less virulent with respect to guinea pigs and chick embryos. The study of the spectinomycin resistant mutants of Legionella spp. did not reveal any changes in the virulence which made it possible to suggest that the influence of the mutations in the ribosomal protein genes determining resistance to streptomycin and neamine on virulence of L. pneumophila was based on the interdependence of the mutant effect on the suppression and the influence on the virulence detected by us in S. flexneri, Y. pseudotuberculosis, L. monocytogenes and F. tularensis. The Legionella mutants Nea(r)Strr100 were characterized by significant protective activity and protected immunized guinea pigs when tested in a model of their aerogenic infection.

Effect of PANTA on growth of Mycobacterium kansasii in BACTEC 12B medium.

Mycobacterium kansasii isolates from two patients showed relatively slow growth in BACTEC 12B medium (12B) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) compared with the more rapid growth of these isolates on solid media. This finding prompted an evaluation of the effect of PANTA (Becton Dickinson) on the growth rate of these isolates. Suspensions of one isolate from each of these two patients (A and B), six additional isolates from six other patients (C through H), and one M. kansasii American Type Culture Collection isolate were inoculated into 12B with PANTA, 12B with reconstituting fluid only, and Middlebrook 7H11 agar plates (Remel, Lenexa, Kans.). For the isolates from patients A and B, the average times to detection for 12B with PANTA, 12B with reconstituting fluid, and Middlebrook 7H11 agar were 12.3, 7.4, and 9.0 days, respectively. For the remaining six patient isolates and the American Type Culture Collection strain, the average times to detection for these media were 9.2, 8.1, and 9.6 days. Susceptibility tests performed with the isolates from patients A and B with the individual component antibiotics of PANTA and testing of four of the other isolates with nalidixic acid alone suggested that nalidixic acid exerts some degree of inhibition on the growth of M. kansasii. The eight patient isolates were also inoculated onto Lowenstein Jensen medium (Remel) and onto a variety of selective mycobacterial media containing nalidixic acid and other antimicrobial agents. All isolates showed some degree of inhibition on at least one of these selective media.

The elimination of Salmonella typhimurium in coastal waters with various levels of microbiologically hygienic contamination.

The biotic elimination of Salmonella typhimurium in coastal sea water is primarily caused by protozoa. The elimination is usually faster in summer than in winter and in the vicinity of waste water outlets partially faster than in coastal areas with less contamination. When the rate of elimination is measured twice in succession in the same sample (primary/secondary culture) the second reduction is considerably faster. This activation is attributed to the multiplication of protozoa (predator-prey-effect). The activation is also possible through E. coli in concentrations such as those found in waste from sewage treatment plants or by Salmonella typhimurium themselves and vice versa. After 12 hours incubation the number of E. coli in the primary culture was still about 58% of the original quantity and 12 hours after a renewed inoculation in the secondary culture only 1%. When salmonella were added to the primary culture it was already impossible to detect E. coli after 12 hours in the secondary culture. Salmonella showed comparable tendencies, although the elimination of salmonella was clearly slower than the elimination of E. coli even after activation with salmonella. In the primary culture E. coli is already recultivatable in a smaller quantity than salmonella. Furthermore the addition of Cycloheximide to the secondary culture provides a considerably better protection for salmonella than for E. coli, so that it can be assumed that other or additional factors are involved in the elimination of E. coli.

Time-survival studies for quantifying effects of azlocillin and tobramycin on Pseudomonas aeruginosa.

Time-survival studies were conducted to estimate the effects of azlocillin and tobramycin on Pseudomonas aeruginosa NCIMB 8295 (in the exponential phase of growth) at concentrations ranging from one-quarter to twice the MIC. The effects of the individual agents and their combinations were determined by measuring the viable counts (CFU per milliliter) over a 24-h period. The typical pattern observed from the plot of the logarithm of the CFU per milliliter against time was an initial rapid killing; this was followed by a period of stasis and regrowth. Initial rates of killing by tobramycin were concentration dependent, whereas this was not the case with azlocillin. From the time-survivor plots, the area under the curve for viable bacteria was also calculated. It offered a useful method of interpreting the results of time-kill studies, taking the overall pattern of killing and regrowth into consideration. The area under the curve for viable bacteria was concentration dependent for both antibiotics. A 2(2) factorial experimental design was used to analyze the joint effects of azlocillin and tobramycin. In such a factorial experiment, an interaction between two factors, in this case, azlocillin and tobramycin concentrations, is shown by a change in the slope of the plot when the concentration of the interactant is changed. Analysis of variance showed that the combination was synergistic at low concentrations, but this was not significant when the concentration of either interactant was increased.