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Antimicrobial peptides from human platelets. Competing interests: The authors have declared that no competing interests exist. The incidence of bacterial bloodstream infections BSI in high-income countries is as extensive as that of strokes, ranging from to cases per , inhabitants [ 1 ].
They are also a leading cause of healthcare-associated infections in intensive care units ICUs [ 3 ] and neonatal wards [ 4 ], and are particularly prevalent in elderly patients 6, cases per , population. Many different bacterial species can cause BSI, among which Escherichia coli , Staphylococcus aureus , Klebsiella species, Pseudomonas aeruginosa , enterococci , streptococci and coagulase-negative staphylococci are the most prominent [ 1 ].
In addition to BSI, it is responsible for a number of life-threatening complications including acute pneumonia and skin infection in immunocompromised and elderly patients, as well as degradation of lung function in chronically-infected cystic fibrosis patients [ 11 ]. The major health concerns related to P.
The capacity of P. Regardless of the primary site of infection, P. In the blood, the bacteria encounter the innate immune system, composed essentially of neutrophils, monocytes, and the complement system. Interactions between P. Recent data indicated that systemic P. However, the mechanisms allowing bacteria to persist in the blood remained unclear.
Within this study, we examined the behavior of a number of laboratory and recently isolated clinical P. Our results showed that, although complement exerts an essential antibacterial activity in the blood, individual bacterial strains display variable levels of tolerance. We evidenced, even for the most sensitive strains, the characteristic biphasic killing curves reminiscent of antibiotic persisters, and characterized a small subpopulation of phenotypic variants that we named complement evaders.
These rare cells withstand complement-mediated lysis through phenotypic heterogeneity. Moreover, we discovered that several other major Gram-negative human pathogens shared the same capacity to escape human complement by forming intrinsically plasma-resistant evaders.
Complement evaders may have a very significant impact on bacterial dissemination. Using a highly standardized method in human whole blood HWB , we examined the survival of six P. In addition to these laboratory strains, we included in the survey three P. Bacteria were incubated for 3 h in HWB from healthy donors, and bacterial survival was assessed in ten independent experiments by counting colony-forming units CFU Fig 1A. Strains showed clearly distinguishable and reproducible survival rates.
Only the non-pathogenic laboratory strain E. In addition, survival in HWB did not correlate with a given serotype, as highly variable survival rates were measured for the three O12 strains Fig 1B. Lack of the O antigen was detrimental for the bacteria, as illustrated by the hypersensitivity of E. When not stated, differences in survival were significant. Dots reported on the x-axis correspond to no detectable colonies.
B Nature of the toxins secreted by each strain, and serotypes of these bacteria. To explore the origin of the extensive differences in survival measured in HWB, we first determined how well each strain was recognized by immune cells. The toxins ExoS, ExoU, and Exolysin A are known to induce apoptosis or necrosis in a variety of eukaryotic cells, including white blood cells which may play a role in bacterial clearance from the blood [ 16 , 31 — 34 ].
We therefore examined the cytotoxic potential of each strain toward purified circulating leukocytes by monitoring lactate dehydrogenase LDH release. YIK induced a complete elimination of neutrophils, whereas most other strains showed similar levels of cytotoxic potential with no significant differences S2A Fig.
Every tested strain appears more efficient at killing neutrophils than mononucleated cells S2B Fig. Thus, except for YIK, the extent to which the bacterial strains tested here were recognized by and destroyed circulating leukocytes could not explain the different survival rates measured in HWB. We next assessed the capacity of the strains to cope with bactericidal effectors present in plasma Fig 2A. As with bacterial survival in HWB, in plasma the survival rates for the six selected P.
The similar survival profiles between HWB and plasma, and the fact that survival of the plasma-sensitive strains CLJ1, IHMA87, and PA14 was fold lower in plasma than in whole blood suggest an important role for humoral immune effectors in bacterial clearance within HWB.
Due to its complete resistance to killing, the YIK strain was excluded from subsequent experiments. A Survival of P. Note similarities in survival profiles to those shown in Fig 1. B Heat-treating plasma prevents elimination of bacteria from HWB. The effect of this treatment on bacterial survival was assessed based on CFU counts.
D Phagocytes are involved in the elimination of a limited number of strains. Dots on the x-axis correspond to no detectable colonies. To assess how fluid-phase effectors contribute to bacterial elimination in HWB, we examined the survival of the different strains by counting CFUs following incubation in HWB reconstituted after heat-inactivation of the plasma see Methods.
Heat-inactivation, which eradicates complement activity, resulted in full survival of all strains, including the sensitive E. Indeed, most strains multiplied when incubated in blood with heat-inactivated plasma, as observed by the increased CFUs compared to the starting population. As heat-inactivation of plasma may inhibit different bactericidal plasmatic components [ 37 ], and to address whether the elimination of sensitive strains in HWB specifically relies on the complement system or other circulating effectors with antimicrobial properties, we used specific complement inhibitor OmCI also known as Coversine [ 38 ].
OmCI inhibits the cleavage of C5, hampering membrane attack complex MAC formation and subsequent complement-mediated lysis [ 37 ]. Addition of OmCI in plasma resulted in complete rescue of P. To elucidate which of these mechanisms was involved in bacterial clearance, we treated the blood with Cytochalasin D to inactivate phagocytosis, or with DNase I to prevent NETs formation [ 22 , 41 ] and monitored bacterial survival.
In contrast, PA7 and PAO1 elimination appear to involve some internalization by phagocytes, as their survival was consistently increased in Cytochalasin D-treated blood Fig 2D. Because the clearance of these two strains was also complement-dependent, we conclude that they are eliminated through opsonophagocytosis. The level of residual surviving cells appears higher by 1-log in whole blood than in plasma Figs 1A and 2A. This higher clearance of bacteria in plasma than in HWB was already observed in the case of Salmonella typhimurium [ 42 ].
A possible explanation could be that within HWB, complement activity is more strongly repressed due to all membrane-bound negative regulators at the surface of circulating leukocytes i.
As indicated above, only the laboratory strain E. The subpopulation of P. We further investigated this intriguing difference in sensitivities using the three complement-sensitive strains PA14, CLJ1, and IHMA87, by carefully examining the kinetics of bactericidal activity in plasma over a 6-h incubation Fig 3A.
Following this first phase, killing slowed down, reached a plateau and left a minor subpopulation of surviving cells. However, this subpopulation failed to grow, even after 6 h. We verified that the drop off in killing rate was not due to depletion of complement activity after 2 h by retesting the used plasma.
Thus, the plasma had a residual bactericidal capacity, sufficient to eliminate a population at least 4-log more numerous than the number of evaders. As further evidence that evaders are not simply a result of bacterial overload of the complement system, inoculating plasma with fold fewer bacterial cells resulted in the same proportion of evaders S3B Fig. Based on these results, evaders correspond to phenotypic variants displaying complement tolerance, and appear to be present in similar proportions to antibiotic-tolerant persisters [ 43 , 44 ].
A Prolonged exposure of P. The three complement-sensitive strains PA14, CLJ1 and IHMA87 were incubated for 6 h in a pool of human plasma, and their survival was measured every 45 min to determine the kinetics of their survival. B The evader phenotype is reversible and not the result of fixed mutations.
Survival was compared to that of a population that was never exposed to complement square. C Evaders are a common feature among Gram-negative bacteria. D The reversibility of the tolerant phenotype was assessed in individual colonies as in B. Note that for K. To further phenotypically characterize evaders, we re-cultured the survivors recovered from a first incubation in plasma and challenged their progeny.
As shown in Fig 3B , following re-culture, the bacterial population had a similar sensitivity profile to previously unchallenged cells. In some cases, the number of evaders in these repeat challenges was below the limit of detection in our experimental settings e. CLJ1 after 2 h. This apparent concern is a hallmark in antibiotic persisters research [ 45 ].
As complement evaders were detected for all sensitive P. To that aim, we selected strains of seven Gram-negative species S1 Table and assessed their survival in pooled plasma. Among the strains tested, Stenotrophomonas maltophilia was undetectable after 1 h of incubation, and Serratia marcescens presented what we called a tolerant phenotype in plasma, with a slow but constant elimination rate.
In contrast, the five other strains tested— Acinetobacter baumannii , Burkholderia multivorans , enteroaggregative EA E. When individual evader colonies were re-cultured, a population as sensitive as the parental one was recovered, as seen for P.
Unexpectedly, for K. Thus, plasma-resistant mutants can be selected and we do not currently know whether selection occurs over the course of the pre-challenge culture steps, or during contact with plasma. As with P. Prevention of MAC formation abolished elimination of A. However, their overall survival was still below 0. We conclude that depending of the strain, plasma evaders are either exclusively or only partly composed of complement-tolerant bacteria, the later observation suggesting that other fluid-phase effectors can be involved in the selection of this subpopulation.
To further describe the features of complement evaders, we assessed if these rare bacteria could arise in conditions known to trigger the emergence of antibiotic-tolerant persisters.
Thus, after verifying bacterial growth-rates and states log versus stationary S5 Fig , we tested whether the evaders observed in exponentially growing cultures corresponded to residual non-growing cells from the previous overnight culture, or to rare cells that had already entered the stationary state after a few hours of culture.
To eliminate possible artefacts due to the growth phases [ 45 , 50 ], we challenged stationary-phase cells or exponentially growing cells with plasma. For the three strains P. Therefore, exponentially growing bacteria had a similar or higher capacity to produce evaders, suggesting that the emergence of evaders is unrelated to dormancy before complement challenge.
To assess whether metabolic shut-down could increase the proportion of evaders, prior to exposure to plasma, P. The addition of CCCP had no effect on bacterial survival in LB S6 Fig , but its use before the plasma challenge completely abolished the detection of evaders after 2 h of incubation Fig 4B , suggesting that proton-motive force is necessary for survival.
Our results show that evaders immediately and actively grow following plasma removal and spotting on agarose pads, forming exponentially-growing microcolonies Fig 4C. In both normal and heat-inactivated plasma-treated conditions, growth rate was low upon spotting 0. Division was not impaired in evaders as the mean time to reach a first division did not vary between normal 1.
Altogether, these results suggest that survival to plasma treatment is not dependent on growth rate but requires energy and active metabolism. A Cells in the stationary phase do not form more evaders than exponentially growing bacteria.
Bacteria from stationary phase and exponentially growing cultures from every evader-forming strain were challenged in a pool of plasma for 3 h to compare their ability to form evaders.
B Formation of evaders requires active metabolism. As controls, bacteria in LB were either not treated or incubated 1 h with 0. C to E Following stress removal, evaders show no growth defect. Using time-lapse microscopy, individual microcolony surface C , mean growth rate D and elapsed time before the first division E were determined.
From two independent experiments, 16 evader cells were analyzed and compared with 16 non-evader cells from the control condition heat-inactivated plasma.
F Time-lapse microscopy of two representative microcolonies from each condition. As we had demonstrated that the central driver of bacterial clearance from blood was the complement system, mainly through its direct lytic activity, we next investigated survival in plasma of a cohort of twelve clinical strains isolated from patients with BSI S1 and S2 Tables to determine their capacity to form evaders.
Like the data obtained with the initial six selected strains, BSI isolates displayed differences in survival rates in plasma, of up to five orders of magnitude.
The limited number of strains and the high diversity of serotypes identified S1 Table would make any attempt to correlate bacterial survival in plasma with strain serotype too speculative. Even though some strains were highly sensitive to plasma, none was fully eliminated. To verify that these surviving cells corresponded to evaders, we also assessed their survival kinetics in plasma Fig 5B. The three isolates PaG3, 9, and 17 presented a tolerant phenotype, with a constant rate of elimination, never reaching a plateau even after 4 h incubation.
In contrast, a biphasic killing curve was recorded for the five other isolates, the kinetics of the curve varied from strain to strain, sometimes reaching a plateau after just 1 h, whereas for others the death rate started to slow from the 3-h time point. As indicated above, in some cases, surviving cells were scarce and bellow the limit of detection e.
PaG8 at 2 and 3 h. Thus, most clinical isolates form evaders that can withstand the bactericidal property of plasma, suggesting that this phenomenon could be exploited by P. A Complement-resistance is not common to all BSI isolates.
Twelve P. B Most complement-sensitive isolates form evaders. Kinetics of survival in pooled plasma for 3 h of eight complement-sensitive clinical isolates.
Three different panels are used for clarity and to allow better visualization of the inflection points. Bacteremia caused by the multi-drug resistant opportunistic pathogen P.
To gain more knowledge on host and pathogen strategies associated with BSI, we undertook an extensive analysis of a cohort of P. Although many previous studies have addressed bacterial transmigration across epithelial and endothelial barriers, the interplay between the immune system and bacterial survival in HWB has been less extensively documented.
Current Surgical Therapy. Philadelphia, PA: Elsevier; Updated by: David C. Editorial team. Bloodborne pathogens. These viruses cause infections and liver damage. HIV human immunodeficiency virus. Sometimes, there are no symptoms. Hepatitis B often gets better on its own and sometimes does not need to be treated. Some people develop a long-term infection that leads to liver damage.
Most people who become infected with hepatitis C develop a long-term infection. After many years, they often have liver damage. Treatment can help people with all of these infections. Hepatitis B can be prevented by a vaccine. There is no vaccine to prevent hepatitis C or HIV. What to Do If You are Exposed.
If you are stuck with a needle , get blood in your eye, or are exposed to any bloodborne pathogen: Wash the area.
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