Plaquing Efficiency Research Articles

The initiation and early development of apical-basal polarity in Toxoplasma gondii.

The human parasite Toxoplasma gondii has a distinctive body plan with a well-defined polarity. In the apical complex, the minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity axis is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex in nascent daughters using expansion microscopy. We found that different substructures in the apical polar ring have different sensitivity to stress. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the construction of the daughter framework progresses towards the centrioles, the apical and the basal arcs co-develop in striking synchrony ahead of the microtubule array, and close into a ring-form before all the microtubules are nucleated. We also found that two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of each protein individually has modest to no impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

High-Throughput Bacteriophage Testing with Potency Determination: Validation of an Automated Pipetting and Phage Drop-Off Method.

The development of bacteriophages (phages) as active pharmaceutical ingredients for the treatment of patients is on its way and regulatory agencies are calling for reliable methods to assess phage potency. As the number of phage banks is increasing, so is the number of phages that need to be tested to identify therapeutic candidates. Currently, assessment of phage potency on a semi-solid medium to observe plaque-forming units is unavoidable and proves to be labor intensive when considering dozens of phage candidates. Here, we present a method based on automated pipetting and phage drop-off performed by a liquid-handling robot, allowing high-throughput testing and phage potency determination (based on phage titer and efficiency of plaquing). Ten phages were tested, individually and assembled into one cocktail, against 126 Escherichia coli strains. This automated method was compared to the reference one (manual assay) and validated in terms of reproducibility and concordance (ratio of results according to the Bland and Altman method: 0.99; Lin's concordance correlation coefficient: 0.86). We found that coefficients of variation were lower with automated pipetting (mean CV: 13.3% vs. 24.5%). Beyond speeding up the process of phage screening, this method could be used to standardize phage potency evaluation.

Evolved distal tail protein of skunaviruses facilitates adsorption to exopolysaccharide-encoding lactococci.

Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures. Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed. Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination. Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS+ host than wild type. Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a subset of skunaviruses.

Cell wall modifications that alter the exolytic activity of lactococcal phage endolysins have little impact on phage growth.

Bacteriophages are a nuisance in the production of fermented dairy products driven by starter bacteria and strategies to reduce the risk of phage infection are permanently sought. Bearing in mind that the bacterial cell wall plays a pivotal role in host recognition and lysis, our goal was to elucidate to which extent modifications in the cell wall may alter endolysin activity and influence the outcome of phage infection in Lactococcus. Three lactococcal endolysins with distinct catalytic domains (CHAP, amidase and lysozyme) from phages 1,358, p2 and c2 respectively, were purified and their exolytic activity was tested against lactococcal mutants either overexpressing or lacking genes involved in the cell envelope stress (CES) response or in modifying peptidoglycan (PG) composition. After recombinant production in E. coli, Lys1358 (CHAP) and LysC2 (muramidase) were able to lyse lactococcal cells in turbidity reduction assays, but no activity of LysP2 was detected. The degree of PG acetylation, namely C6-O-acetylation and de-N-acetylation influenced the exolytic activity, being LysC2 more active against cells depleted of the PG deacetylase PgdA and the O-acetyl transferase OatA. On the contrary, both endolysins showed reduced activity on cells with an induced CES response. By measuring several growth parameters of phage c2 on these lactococcal mutants (lytic score, efficiency of plaquing, plaque size and one-step curves), a direct link between the exolytic activity of its endolysin and phage performance could not be stablished.

Comparing patient-centered outcomes and efficiency of space closure between nickel-titanium closed-coil springs and elastomeric power chains during orthodontic treatment.

To compare patient-reported pain, discomfort, and difficulty in maintaining proper brushing between nickel-titanium closed-coil springs (CS) and elastomeric power chains (PC) when used for space closure. The secondary aims were to compare plaque control and efficiency of space closure between these two force delivery systems. A total of 48 patients who required extractions of upper first premolars and distal movement of upper canines had the CS randomly allocated to either the right or left side. Blinding was applied at data collection and analysis. Primary outcomes were pain intensity measured on visual analog scale, pain onset and duration, discomfort, and difficulty in maintaining proper brushing from the start of canine retraction at baseline and at 6 and 12 weeks thereafter. Secondary outcomes were plaque scores and the rate of space closure. No significant differences in mean pain scores, pain onset, and duration at different time intervals between CS and PC were observed. The CS side was significantly less comfortable than the PC (P < .0001) and more difficult to keep clean (P = .008). No significant differences in plaque scores were observed between CS and PC groups at any time interval. CS produced a faster rate of space closure than did PC (P = .008). CS were less tolerated than PC by patients but produced an average of 0.5 mm more movement than did the PC during the 12-week study period.

RNA-seq reveals the phage-resistant mechanisms displayed by Lactiplantibacillus plantarum ZJU-1 isolated from Chinese traditional sourdough

Seven spontaneous phage-resistant mutants were obtained from a wild-type Lactiplantibacillus plantarum. Phage-resistance stability, efficiency of plaquing (EOP), adsorption rates, lysogeny, growth, and acid production of mutants B4 and B7 were determined. The phage defence mechanism of B4 was revealed by RNA-seq. Both B4 and B7 exhibited very low EOP values and high phage-resistance stabilities. Adsorption rates of B4 and B7 in MRS-Ca broth were diminished to 69.14% and 97.96%, respectively. Technological properties in the presence of phages were similar to the wild-type Lb. plantarum. RNA-seq revealed that genes of B4 were differentially expressed when compared with the wild-type Lb. plantarum. Genes involving in cell surface proteins, phosphoenolypyruvate sugar phosphotransferase system, and EPS production were up-regulated. The phage-resistance of B4 was increased by the downregulation of genes coding for prophages and prophage-induced lysins proteins. The results give clues for a novel strategy of controlling phage contamination in dairy industry.

Identification of a Prophage-encoded Abortive Infection System in Levilactobacillus brevis

Abortive infection systems (Abi) are phage resistance systems that can be prophage-encoded. Here, two genes encoding an Abi system were identified on a prophage sequence contained by the chromosome of the Levilactobacillus brevis strain UCCLBBS124. This Abi system is similar to the two-component AbiL system encoded by Lactococcus lactis biovar. diacetylactis LD10-1. The UCCLBBS124 prophage-derived Abi system (designated here as AbiL124) was shown to exhibit specific activity against phages infecting L. brevis and L. lactis strains. Expression of the AbiL124 system was shown to cause reduction in the efficiency of plaquing and cell lysis delay for phages of both species.

Cyanophage Propagation in the Freshwater Cyanobacterium Phormidium Is Constrained by Phosphorus Limitation and Enhanced by Elevated pCO2.

Intensification of human activities has led to changes in the availabilities of CO2 and nutrients in freshwater ecosystems, which may greatly alter the physiological status of phytoplankton. Viruses require hosts for their reproduction and shifts in phytoplankton host physiology through global environmental change may thus affect viral infections as well. Various studies have investigated the impacts of single environmental factors on phytoplankton virus propagation, yet little is known about the impacts of multiple factors, particularly in freshwater systems. We therefore tested the combined effects of phosphorus limitation and elevated pCO2 on the propagation of a cyanophage infecting a freshwater cyanobacterium. To this end, we cultured Phormidium in P-limited chemostats under ambient (400 μatm) and elevated (800 μatm) pCO2 at growth rates of 0.6, 0.3, and 0.05 d-1. Host C:P ratios generally increased with strengthened P-limitation and with elevated pCO2. Upon host steady state conditions, virus growth characteristics were obtained in separate infection assays where hosts were infected by the double-stranded DNA cyanophage PP. Severe P-limitation (host growth 0.05 d-1) led to a 85% decrease in cyanophage production rate and a 73% decrease in burst size compared to the 0.6 d-1 grown P-limited cultures. Elevated pCO2 induced a 96% increase in cyanophage production rate and a 57% increase in burst size, as well as an 85% shorter latent period as compared to ambient pCO2 at the different host growth rates. In addition, elevated pCO2 caused a decrease in the plaquing efficiency and an increase in the abortion percentage for the 0.05 d-1 P-limited treatment, while the plaquing efficiency increased for the 0.6 d-1 P-limited cultures. Together, our results demonstrate interactive effects of elevated pCO2 and P-limitation on cyanophage propagation, and show that viral propagation is generally constrained by P-limitation but enhanced with elevated pCO2. Our findings indicate that global change will likely have a severe impact on virus growth characteristics and thereby on the control of cyanobacterial hosts in freshwater ecosystems.

Isolation and characterization of spontaneous phage-resistant mutants of Lactobacillus paracasei

Spontaneous phage-resistant mutants were isolated from Lactobacillus paracasei LPC by agar plate (AP) and secondary-culture (SC) methods. They were characterized by cell and colony morphologies, carbohydrate fermentation patterns, phage resistance stability, efficiency of plaquing (EOP), acidifying and milk acidification kinetics. Only 98 out of 175 isolates (56%) proved to be true phage-resistant mutants and the SC method was more efficient than the AP method. Although the phage resistance stability varied among the mutants isolated, the EOP values were mostly very high (<10−11). Three phage-resistant strains were selected based on their extreme resistance capacity to the phage ΦT25 (EOP<10−10) and their nonlysogenic property. Acidifying activity did not differ between phage-sensitive strains and their three respective phage-resistant derivatives. Most of the mutants were completely or partially unable to adsorb phage particles. Restriction-modification type systems were not detected in all phage-resistant derivatives. All three selected mutants were identical to the corresponding parent strain of L. paracasei LPC in the cell and colony morphologies. The comparison of random amplified polymorphic DNA profiles obtained with four arbitrary primers also revealed the highest similarity coefficient (87%) among the parent strain and the three mutants, indicating that each mutant has been derived from this parent strain. A good performance during milk fermentation and the subsequent refrigerated storage were obtained when these mutants were employed. These phage-resistant derivatives could be used as improved strains or for strain rotation programs when commercial strains become sensitive to the phages present in industrial environments.

Technological performance of spontaneous phage-resistant derivatives of Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides during milk fermentation

A total of 142 presumptive phage-resistant derivatives were isolated from six commercial phage-sensitive Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides strains, using six phages either individually or in combination (cocktail). Genetic diversity, efficiency of plaquing (EOP), phage-resistance stability, lysogeny, adsorption rates and technological performance were determined for the 89 confirmed phage-resistant variants. Derivatives showed very low EOP values (<10−10) and high stability of phage-resistance phenotype. Some mutants showed low adsorption rates (10–42%) thus indicating adsorption interferences. Additional resistance mechanisms (operative at later stages) were suggested for mutants revealing high phage adsorption rates. A good performance during milk fermentation and the subsequent refrigerated storage in the presence of phages was demonstrated for four mutants, selected on the basis of their stability and low or null phage adsorption. These phage-resistant variants could be used in industrial rotation schemes when commercial strains become sensitive to phages present in the environment.

Combined Effects of Elevated pCO2 and Warming Facilitate Cyanophage Infections.

Elevated pCO2 and warming are generally expected to influence cyanobacterial growth, and may promote the formation of blooms. Yet, both climate change factors may also influence cyanobacterial mortality by favoring pathogens, such as viruses, which will depend on the ability of the host to adapt. To test this hypothesis, we grew Plectonema boryanum IU597 under two temperature (25 and 29°C) and two pCO2 (400 and 800 μatm) conditions for 1 year, after which all treatments were re-exposed to control conditions for a period of 3 weeks. At several time points during the 1 year period, and upon re-exposure, we measured various infection characteristics of it associated cyanophage PP, including the burst size, latent period, lytic cycle and the efficiency of plaquing (EOP). As expected, elevated pCO2 promoted growth of P. boryanum equally over the 1 year period, but warming did not. Burst size increased in the warm treatment, but decreased in both the elevated pCO2 and combined treatment. The latent period and lytic cycle both became shorter in the elevated pCO2 and higher temperature treatment, and were further reduced by the combined effect of both factors. Efficiency of plaquing (EOP) decreased in the elevated pCO2 treatment, increased in the warm treatment, and increased even stronger in the combined treatment. These findings indicate that elevated pCO2 enhanced the effect of warming, thereby further promoting the virus infection rate. The re-exposure experiments demonstrate adaptation of the host leading to higher biomass build-up with elevated pCO2 over the experimental period, and lower performance upon re-exposure to control conditions. Similarly, virus burst size and EOP increased when given warm adapted host, but were lower as compared to the control when the host was re-exposed to control conditions. Our results demonstrate that adaptation but particularly physiological acclimation to climate change conditions favored viral infections, while limited host plasticity and slow adaptation after re-exposure to control conditions impeded host biomass build-up and viral infections.

Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides bacteriophages: Genomics and cross-species host ranges

Unveiling virus-host interactions are relevant for understanding the biology and evolution of microbes globally, but in particular, it has also a paramount impact on the manufacture of fermented dairy products. In this study, we aim at characterizing phages infecting the commonly used heterofermentative Leuconostoc spp. on the basis of host range patterns and genome analysis. Host range of six Leuconostoc phages was investigated using three methods (efficiency of plaquing, spot and turbidity tests) against Ln. mesenteroides and Ln. pseudomesenteroides strains. Complete genome sequencing from four out of the six studied Leuconostoc phages were obtained in this work, while the remaining two have been sequenced previously. According to our results, cross-species host specificity was demonstrated, as all phages tested were capable of infecting both Ln. pseudomesenteroides and Ln. mesenteroides strains, although with different efficiency of plaquing (EOP). Phage adsorption rates and ability of low-EOP host strains to propagate phages by crossing the Leuconostoc species' barrier confirm results. At the genome level, phages CHA, CHB, Ln-7, Ln-8 and Ln-9 revealed high similarity with previously characterized phages infecting mostly Ln. mesenteroides strains, while phage LDG was highly similar to phages infecting Ln. pseudomesenteroides. Additionally, correlation between receptor binding protein (RBP) and host range patterns allowed us to unveil a finer clustering of Leuconostoc phages studied into four groups. This is the first report of overlapped phage host ranges between Leuconostoc species.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems.

Temperature Significantly Affects the Plaquing and Adsorption Efficiencies of Listeria Phages.

Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria) against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b) grown under a range of temperatures (7–37°C). We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048’s putative phage receptor) on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37°C, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids (WTA) than the parental strain at both 30 and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the WTA. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications.

The Clostridium difficile cell wall protein CwpV confers phase-variable phage resistance.

SummaryBacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. C lostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell‐surface protein CwpV provides antiphage protection in C . difficile. This protein, for which the expression is phase‐variable, is classified into five types, each differing in their repeat‐containing C‐terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C . difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10−7 for phages ϕCD38‐2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10−3 and 1.1 × 10−1. The C‐terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host‐encoded and phase‐variable antiphage system in C . difficile.

Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ.

The lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage of antiQ by ABIQ was characterized using 5' rapid amplification of cDNA ends PCR and was located in an adenine-rich region of antiQ. We next generated a series of derivatives with point mutations within antiQ or with various numbers of antiQ repetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number of antiQ repeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile of antiQ. Interestingly, a point mutation in the putative pseudoknot structure of antiQ mutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.

Isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against Salmonella enterica serovars

The use of bacteriophages is considered as a viable alternative to chemical antimicrobials against foodborne pathogens. The objective of this study was to develop a collection of lytic bacteriophages which will be able to infect different pathogenic Salmonella enterica serovars. Phages were isolated from animal feces and sewage samples, purified, characterized morphologically and by DNA fingerprinting, and host ranges were determined. Spot test and efficiency of plaquing (EOP) data indicated that two phages, SEA1 and SEA2 had the broadest host range against Salmonella among all isolated phages. SEA2 was highly efficient to infect S. Typhimurium DT104 (0.5–1 EOP value). Only phage SSA1 was able to infect S. Montevideo. Transmission electron microscopy (TEM) revealed the phages in the collection were mostly (4 out of 6) Siphoviridae, while SEA1 and SEA2 were Myoviridae T4-like phages. SEA1 and SEA2 had the largest genome sizes in the collection, 190 and 170 kb, respectively. Pulsed field gel electrophoresis (PFGE) analysis demonstrated distinct digestion profiles with EcoRI for phages SSA1, STD3, STE3 and STF1. However, SEA1 and SEA2 shared a similar restriction enzyme (RE) digestion pattern with same morphotype, but distinct profiles in lysing Salmonella strains. These anti-Salmonella phages were highly host specific with few exceptions of lytic phages that were able to infect a wide variety of Salmonella. These phages have potential for use in applications controlling Salmonella on different matrices.

Phase Variable Expression of Capsular Polysaccharide Modifications Allows Campylobacter jejuni to Avoid Bacteriophage Infection in Chickens

Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage–host co-evolution.

Comparative evaluation of the sensitivity of Acinetobacter to colistin, using the prediffusion and minimum inhibitory concentration methods: detection of heteroresistant isolates

The objective of this study is to perform a comparative evaluation of the prediffusion and minimum inhibitory concentration (MIC) methods for the detection of sensitivity to colistin, and to detect Acinetobacter baumanii-calcoaceticus complex (ABC) heteroresistant isolates to colistin. We studied 75 isolates of ABC recovered from clinically significant samples obtained from various centers. Sensitivity to colistin was determined by prediffusion as well as by MIC. All the isolates were sensitive to colistin, with MIC = 2µg/ml. The results were analyzed by dispersion graph and linear regression analysis, revealing that the prediffusion method did not correlate with the MIC values for isolates sensitive to colistin (r² = 0.2017). Detection of heteroresistance to colistin was determined by plaque efficiency of all the isolates with the same initial MICs of 2, 1, and 0.5 µg/ml, which resulted in 14 of them with a greater than 8-fold increase in the MIC in some cases. When the sensitivity of these resistant colonies was determined by prediffusion, the resulting dispersion graph and linear regression analysis yielded an r² = 0.604, which revealed a correlation between the methodologies used.

A Single Mutation Responsible for Temperature-Sensitive Entry and Assembly Defects in the VP1-2 Protein of Herpes Simplex Virus

Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.