We also assessed the effects of past treatments on bacterial virulence in vivo, employing larvae of Galleria mellonella to survey the treatment consequences for the pathogen. We find a strong synergistic effect of combining antibiotics and phages on bacterial population density and in limiting their recovery rate. Our long‐term study establishes that antibiotic dose is important, but that effects are relatively insensitive to antibiotic type. From an applied perspective, our results indicate that phages can contribute to managing antibiotic resistance levels, with limited consequences for the evolution of bacterial virulence.With escalating resistance to antibiotics, there is an urgent need to develop alternative therapies against bacterial pathogens and pests. One of the most promising is the employment of bacteriophages (phages), which may be highly specific and evolve to counter antiphage resistance. Despite an increased understanding of how phages interact with bacteria, we know very little about how their interactions may be modified in antibiotic environments and, reciprocally, how phage may affect the evolution of antibiotic resistance. We experimentally evaluated the impacts of single and combined applications of antibiotics (different doses and different types) and phages on  Despite widespread bacterial resistance and dwindling discovery of new molecules, antibiotics are still overwhelmingly the principal agent used against bacterial infections   Kirby , PseudomPseudomonas aeruginosa, is a leading cause of nosocomial infections and chronic lung infections in patients with cystic fibrosis  and used as the ancestral phage for all the experiments.We used the bacterium P. aeruginosa PAO1 were isolated in KB solid agar plates from the clonal stock stored at −80°C. Three microcosms with 6 mL of KB were used to each amplify a different, arbitrarily selected clone under continuous shaking (200 rpm) for 6 h to obtain an exponential phase culture of c 106 cells/mL. These microcosms were then mixed and used as the inoculum for the evolution experiment.Individual clones of c 2 × 103 cells of P. aeruginosa PAO1 (from a mix of the three microcosms) were inoculated into fresh media containing phage‐only, antibiotic‐only, or phage‐antibiotic conditions. We used the antibiotics carbenicillin, gentamicin, and trimethoprim , belonging to the following families : β‐lactam ; aminoglycoside (blocks protein synthesis); sulfamide (interferes with nucleic acid synthesis), respectively. These antibiotics were added to liquid medium at the required concentrations to inhibit 5%, 50%, and 95% of bacterial growth, hereafter referred to as IC5, IC50, and IC95, respectively ]. An equivalent number of replicates per plate with phage‐only treatments and nontreated bacteria (positive controls) were also established (six replicates × two controls × three plates: 36 populations). The total number of populations was 144. Negative controls with media only were distributed in all plates to monitor for the occurrence of possible contamination.Bacterial density was measured every 24 h by means of optical density (OD) at 600 nm . So as to homogenize cultures, thorough pipette mixing of each well was performed before transferring each population into a new well containing either fresh media under the same treatment conditions  or under control conditions. A volume corresponding to 10% of each population was calculated and transferred, to minimize possible bottlenecks. This same protocol was carried out for seven consecutive days. After being transferred, the remainder of the microcosm was stored in 20% glycerol at −80°C for further analysis.g, the supernatant discarded, and the pellet resuspended in M9. This procedure eliminated most previous phage present in the evolved bacteria. OD was then standardized to the same level for all tested populations, c 106 cells added to 200 μL of KB with and without the ancestral LKD16 phage (1 PFU/100 bacteria), and OD recorded every 20 min for 24 h at 37°C. To minimize the effects of condensation and biofilm interference on OD readings, 10 s of agitation at 200 rpm was programmed to occur before each measurement.To measure bacterial resistance to phage, we compared the growth of either evolved or ancestral bacteria, with or without ancestral phage. Final bacterial populations and the ancestral stock, all stored at −80°C, were streaked on solid agar KB plates to isolate colonies. Four replicate populations of each treatment were used, and one colony of each was amplified in 1 mL of KB in 48‐well plates at 37°C. After overnight incubation, all cultures were centrifuged at 20 000 c 2 × 103 cells of each final treatment population were inoculated into 200 μL of media containing concentrations of each respective antibiotic at twofold increments. After 24 h, we scored growth inhibition as OD < 0.1. Antibiotic resistance was scored for the evolved populations relative to that of the ancestor . Thus, a value of two reflects a doubling in the MIC relative to the ancestral clone, three is twice the MIC of two, and four twice the MIC of three.Antibiotic resistance level was measured as the minimum inhibitory concentration (MIC) at which no bacterial growth was detected. Approximately c 5 × 103 cells/mL. 20 μL of each bacterial treatment was injected into each of 12 wax moth  larvae of homogeneous size (c 2–3 cm) with a micro‐injector. Negative control treatments injecting only M9 were also performed. Larvae were kept at 37°C. The virulence of evolved and ancestral bacteria was assessed as mortality rate per treatment, established by observing larval mortality every hour for 48 h.The virulence of evolved bacteria and ancestral clones was measured by inoculating a sample from each of the frozen final populations and the frozen ancestral stock, respectively, into fresh media and incubating at 37°C until obtaining exponential phase cultures (OD between 0.4 and 0.7). Bacterial replicates from the same treatment were then pooled, centrifuged, and resuspended in M9 buffer and the density adjusted through dilution to http://www.r-project.org/). For the main analysis, we applied fully factorial models by including phage treatment (yes/no), antibiotic type, and antibiotic dose as explanatory factors. Repeated measures through time were included as random factors when present. Where appropriate, analyses were carried out separately for single and combined treatments.All statistical analyses were conducted using R statistical software  densities. To calculate the rate of adaptation over the seven days of the experiment, a nonparametric and nonlinear approach was carried out as follows. First, the deviation from the mean OD of the untreated control of each plate at each time  was subtracted from the observed OD values to correct for ‘plate’ and ‘time’ effects. In a second step, a model was fitted for each replicate via a logistic‐type nonlinear least squares function  from the following equation:anovas to analyze the effect of the different treatments on r.A smoothed cubic spline was used to estimate the lag phase phage + ΔODab < ΔODphage+ab, where Δ is the absolute decrease in optical density (OD), and phage+ab is the combined treatment. The maximum additive effects were limited to the untreated control OD values, the biological meaningful threshold . We then compared densities between observed and expected replicates in a factorial model. A nonparametric Spearman correlation was used to test the linear dependence between antibiotic dose and synergy  variables.We investigated whether combined treatments were subadditive, additive, or superadditive  by pairing random couples of single‐phage with single‐antibiotic replicates at each time‐point of the evolution experiment for each dose and antibiotic type. For both replicates in a pair, we calculated the reduction in bacterial density relative to the untreated controls. We then added the two single treatment reductions to obtain the expected density in a hypothetical combined treatment. We performed this operation for every replicate in the experiment. The effect was scored as synergistic if ΔODanovas. Multinomial analyses were used to analyze the effect of treatments on the final OD.We employed General Linear Model techniques to analyze variation in antibiotic resistance levels (MIC). To study the possibly inhibitory effect of ancestral phages in the final populations, we considered growth over 24 h and the final OD after 24 h. The area under the curve (AUC) was estimated for each OD dynamic with the R package ‘MESS’ Ekstrom . The estTime‐dependent larval survival was estimated using the Kaplan–Meier technique . Specifically, bacteria treated with phage and antibiotics reached an average final optical density (OD) of 0.92 ± 0.07, whereas antibiotic‐only treated bacteria had an OD of 1.32 ± 0.05. Final density values showed a significant negative correlation with antibiotic dose . These results indicate that the addition of phage combined with high doses of any of the antibiotics reduces the long‐term growth of P. aeruginosa PAO1.As expected, the addition of phage or antibiotic had a strong negative effect on bacterial densities during the experiment . On average, slopes calculated with a nonparametric and nonlinear approach  are higher and increase with antibiotic dose  under antibiotic‐only treatments (2.08 ± 0.36) . In contrast, in the double treatments we observe smaller recovery rates (1.85 ± 0.22), higher at the lowest antibiotic doses, and decreasing as the concentration of antibiotic increases, indicating a significant interaction between the addition of phages and antibiotic dose . These results thus show a pronounced effect of high antibiotic dose when combined with phages in limiting bacterial population recovery, whereas bacteria recover faster with high dose of antibiotics alone. Furthermore, we detect significant effects of using different antibiotic types in single treatments, but not in the presence of phages .We find that bacterial density recovery rates  are marginally affected by the addition of phages and significantly by antibiotics . However, we see no such effect over the first 2 days of the experiment . There were significant differences between antibiotic types in the synergistic effect through time , whereas the antibiotic dose component plays an important role for the duration of the experiment and at the final time‐point  was significantly higher than the expected additive effect . Overall, the combination of phages and antibiotics had a synergistic effect on reductions in bacterial density  populations carried costs that impaired their normal growth. To investigate these possibilities, we measured: (i) ancestral phage effects on bacterial growth capacity, (ii) antibiotic resistance levels, and (iii) growth capacity of the final bacterial populations.To estimate the effect of previous treatment on bacterial growth in the presence of ancestral phages, we isolated colonies from the end of the experiment (day 7) and inoculated them with the ancestral phage. We then measured final densities after 24 h to assess the inhibitory effect of phages and also the area under the curve (AUC) as an estimate of the overall bacterial growth capacity . Surprisingly, this reduction was not significantly different between bacterial populations previously treated with or without phage . These results suggest that, independent of treatment, evolved bacteria were still highly susceptible to the ancestral phage.We observed a strong effect of ancestral phage in decreasing bacterial densities after 24 h . In single treatments, neither antibiotic type nor dose significantly affected the AUC in the presence of ancestral phage . In contrast, antibiotic type was found to be significant in combined treatments, whereas there was no effect of antibiotic dose . However, when combined and only‐phage treatments were analyzed separately, none of the three antibiotics had a significant effect on phage resistance . These results show that treatments with and without phages have different growth dynamics in the presence of the ancestral phage, even though the final outcome  was apparently unchanged, indicating the expression of partial resistance.A deeper analysis of the effect of the ancestral phage on overall 24 h bacterial growth measured as the AUC (area under the curve) revealed slightly different effects. When confronted with ancestral phage, bacteria that had been previously treated with phage showed a linear increase and higher AUC values than non‐phage‐treated bacteria, the latter exhibiting an initial lag phase with a small peak, followed by exponential recovery . We found evidence for an antibiotic type effect, but not an antibiotic dose effect on the MIC increase relative to the ancestral bacteria . These results suggest that phages constrain the evolution of antibiotic resistance in combined treatments.We then tested whether the combined treatments limited the final level of antibiotic resistance (MIC) acquired by the surviving evolved bacteria compared to ancestral levels. We also determined the magnitude of the change in antibiotic resistance generated by the different antibiotic doses and types. Treatments with antibiotics resulted in different levels of increase in the MIC compared to controls, but the addition of phages attenuated this effect  and measuring their life span. Supplementing phages in addition to antibiotics daily in vitro reduced P. aeruginosa final virulence in wax moth larvae compared to nontreated controls, but the reduction was smaller than that of bacteria treated with antibiotics alone , but still significantly less virulent than ancestral bacteria . In general, all evolved bacteria (controls and treatments) were less virulent relative to ancestral bacteria , and we found statistically significant differences depending on the antibiotic type and dose used , with low doses reducing virulence compared to higher doses.We then analyzed the consequences of the 7‐day treatments on bacterial virulence P. aeruginosa.Previous study has considered the separate effects of phages and antibiotics on bacterial population sizes and resistance , simultaneous resistance to antibiotics and phages entails larger costs than to either mortality agent separately. The net result is lower than expected populations in the combined treatments, but only once costly resistance emerges as a result of selection and evolution. Consistent with this explanation, several studies have shown that phages limit the emergence of antibiotic resistant variants in combined treatments , possibly due to resistance mutations in the peptidoglycan synthesis pathway, this being implicated in bacterial pathogenesis (Godlewska et al. Finally, past studies have found different relationships between elevated antibiotic resistance and virulence expression, the most frequent being negative (Beceiro et al. P. aeruginosa, and that other antibiotic‐phage‐bacterial pathogen systems need to be assessed. Regulatory requirements for the employment of phages are challenging (Verbeken et al. Our results provide important insights for the potential use of phage‐antibiotic combinations in controlling bacterial pathogens and pests. The strong bacterial density reduction caused by the combined action of phages and high doses of antibiotics, together with the reduced probability of bacterial resistance evolution under these conditions, support the choice of high doses of antibiotics combined with lytic phages as an evolution‐minimizing approach against bacterial pathogens and pests. We stress, however, that we only employed one phage type and that future research on other phages is needed to assess the generality of our results for this strain of 10.6084/m9.figshare.2065209.All data can be accessed through figshare.com. doi: Figure S1. Bacteria density dynamics measured every 24 h for 7 days.Click here for additional data file.Figure S2. Slopes of bacterial density with and without phages, for each antibiotic dose.Click here for additional data file.Figure S3. Synergistic effects of treatments through time: expected additive versus observed effects of combined phage‐antibiotic treatments in preventing growth in bacterial populations.Click here for additional data file.Figure S4. Probability of survival of Galleria mellonella larvae inoculated with final bacterial populations.Click here for additional data file.


To analyze the genesis of the policy for controlling AIDS in Brazil. Socio-historical study (1981-1989), based on Bordieu’s genetic sociology, by document analysis, bibliographical review, and in-depth interviews. It consisted of a connection between the analysis of the paths of 33 agents involved in the creation of a social space focusing on AIDS-related issues and the historical possibility conditions of the drafting of a specific policy. AIDS Space is a gathering point for the paths of agents from several social fields . A specific space for relationships, which enabled the drafting of a policy for controlling the AIDS epidemic, but also a place where the authority to talk about the meaning of the disease, the methods to prevent and treat it was under dispute. The analysis showed how the various structures  and the lack of a specific therapy contributed to social agents of different ranks and backgrounds to initially set prevention as a priority. The rise of the sanitary movement, the organization of SUS, and the dominance of the medical field at the AIDS Space contributed to foster treatment as a part of the measures to control the epidemic. These conditions allowed drafting a policy based on the integrality of care, by linking prevention and treatment in the following decade, with important participation from state bureaucracy and researchers. Those who analyzed the government response to the epidemic at the national level have not investigated the participation of medical and scientific fields, nor have they considered the social and professional paths of agents18.The studies on the origins of Brazil’s AIDS policy, which were reviewed, focused on analyses of state cases, especially Sao Paulo5 into practice, Pinell et al.22 studied the universe of possibilities for the rise of a movement to fight AIDS in France, its structure, and its dynamics of relationships of AIDS Space agents between 1981 and 1996. This space was created in that country through a popular mobilization that aimed to replace state institutions in the performance of this public role. The policy against AIDS was drafted by the initiatives of militant associations, the advancements in the medical field, and the illness social representation as a threat to French society.By putting the concept of social space developed by Bourdieu17, based on Bordieu’s field concept, connected the position at the “HIV/AIDS field” with entrepreneurial action methods. Their analysis focused on the medical field and on specific associations, but did not explore the interaction between the paths of agents and conditions of possibility. The development of a space for fighting AIDS is understood to depend on the medical knowledge on the disease, but not to fulfill all the requirements of a field, as proposed by Bordieu. The concept of AIDS Space as suggested by Pinell et al.22 is more appropriate because it is a space of relationships among the agents in different social fields.The study by Mendonça et al.The studies reviewed do not shed light on how the AIDS Space was organized in Brazil  nor do they explain why a policy was drafted at a time when there was no evidence on its extent and vulnerability. Neither have they systematically analyzed the liaisons between agents, the points of view or strategies they chose regarding the policy on AIDS, concerning their fields of origin.8.This study on the genesis of Brazil’s AIDS policy provides elements that help fill these gaps, based on Bourdieu’s reflexive sociologyj, considering the main groups affected, times, and priority actions of the government response and its relationship with medical knowledge    and the 25 . Despite the role of the press in giving the first reports on AIDS, only in 1983 were the first Brazilian cases reported2.The first news reports about the disease were treated as a foreign problem by the mediaDespite first denying the disease, the gay movement was fundamental to recognize AIDS as a problem that required specific measures. The questioning stance on medical field was a result from the historical relationship of some physicians with homosexuality, who viewed it as a mental illness.“[...] Militants tended to think that it was another lie from physicans [...]. Yet another way to control homosexuality, to get people to quit these behaviors” .m.Afterwards, this group started reinforcing medical discourse, probably as a result from the exchange with foreign groups who brought reports of fear and death, and also because of the threat to the developing gay trade. [...] I heard about people dying like flies. [...] almost no one believed it” .“[...] I spent a week in New York9.The epidemic transformed the gay rights movement, by causing its sexual freedom proposals to lose momentum and reducing the number of groupsogathered gay activists in March 1983, warning them about the need for a response from the government.Besides diagnosing the first cases, physicians from different backgrounds took part in the initiatives that resulted in the first measures for control of the disease. Ricardo Veronesi ,“[...] From then on, they started getting organized [...] to try to ensure some achievements, and, at the same time, discuss the AIDS issue and push authorities” .24.Motivated by the meeting and by the first AIDS cases in Brazil, Darcy Penteado scheduled an appointment with Sao Paulo’s Health Care Officer, João Yunes“[...] a demand that had been started by people that had already [...] taken part in the gay rights movement. [...] and demanded the [State Health] Secretariat [...] take measures, provide guidance, give information regarding the epidemic [...]” Grupo Somos and of the “Lampião da Esquina” newspaper, such as anthropologist Edward MacRae, Belgian filmmaker Jean Claude Bernadet, writer and journalist João Silvério Trevisan, and physician Valéria Petri , followed Darcy Penteado. They requested that Sao Paulo’s State Heath Secretariat (SES-SP) made a statement about AIDS and future cases treatment. This meeting motivated a movement for creating the AIDS Program at SES-SP, at Hansen’s Disease and Public Health Dermatology Division, which was coordinated by physician Paulo Roberto Teixeira, a former member of Grupo Somos (E26).Former members of 24, the following contributed to the implementation of the AIDS Program at SES-SP: the first cases diagnosed; the demand for a group of militants, with support from a female physican; and the political conditions . The fear of the epidemic, the social group affected , and the fact Sao Paulo had had a health care system with public health officer positions as of the 1960s should be addedp, as well as the profile of the system’s director (a sanitarian and former member of the gay rights movement), a person with technical and political capability of dealing with stigmatized groups (E26).According to other studiesFederal education and research institutions engaged by initiative of their professors and researchers. For example, Escola Paulista de Medicina, by physician Valéria Petri (E31), and Fiocruz, by its AIDS Research Laboratory. The latter was created in 1983, in Rio de Janeiro, by Bernardo Galvão, physician, master in Human Pathology, and PhD in Immunology, who became its coordinator, and Cláudio Ribeiro, a PhD in Immunohematology (E4).The rise of Sao Paulo State AIDS Program confirmed the organization of a space to fight AIDS (AIDS Space). It was also a space for research and intervention, involving agents in the gay rights movement (militant space), in the medical field, teaching professionals, and researchers (scientific field), or staff in the state health care office (bureaucratic field).q. Fabíola de Aguiar Nunes, a sanitarian physician, took part in the program as a representative from the Ministry of Education and Culture (E10).In February 1985, federal government initiatives for controlling AIDS were initiated through Professional Training for Controlling Hospital-Acquired Infections Program, which was coordinated by Luiz Carlos Pelizari Romero, from the National Secretariat for Special Health Care Programs (SNPES)When Carlos Sant’Anna, a physician, congressman, and one of the liaison agents in Tancredo Neves’ presidential election run, took over the Ministry of Health (MH) in March 1985, Fabíola Nunes took over SNPES, where, in the National Public Health Dermatology Division (DNDS), Brazil’s National Program on Sexually-Transmitted Diseases (STD) and AIDS (PNAids) would be created .The first measures taken by the MH were epidemiological surveillance, public health education and information, voluntary testing, and characterization of an epidemic that affected the whole population, to reduce stigma and discrimination of the most affected groups .Several social movements  demanded a response from the government. The press insisted on the issue . Sao Paulo, Rio de Janeiro, and Rio Grande do Sul had already structured measures (E24), pressing the federal government to make a decision. The social representation of the disease, coupled with terror and fear, should be added to this.r. Epidemiological, clinical, and laboratory investigation were proposed, as well as public health education for suspected and confirmed cases, people in contact, and high-risk groups. The ordinance mentioned using condoms, disposable or individual syringes and needles, and controlling blood quality as preventive measuress. A training system was organized in Sao Paulo, for the states reporting their first cases (E10).In March 1985, meetings were held in Sao Paulo and Brasilia, to review and discuss the cases diagnosed. These meetings respectively resulted in the standardization of AIDS management procedures and the drafting of an ordinance by a panel of experts19. Lair Guerra de Macedo Rodrigues, a biomedical and professor at Universidade de Brasilia who had taken a specialization course at the Centers for Disease Control and Prevention (CDC) in the USA, was invited by Maria Leide to take over the AIDS program. The motivations behind this invitation were her experience at the CDC, her good references, and her potential influence and international support, as Carlyle Guerra de Macedo’s, a representative from the Pan American Health Organization (PAHO) was her brother (E24). As Maria Leide joined, a committee on risk group 19 was created. It was the precursor of Brazil’s National Aids Commission (CNAids) (E24) and it played an important technical and political role, by providing guidance to PNAids on the definition of adopted strategiest.In January 1986, Fabíola Nunes invited dermatologist Maria Leide de Oliveira to DNDS . Miriam Franchini was responsible for STDs and sanitarian Lúcia Amaral, for AIDS, organizing an initial reporting system24.The initiatives aimed at structuring the program and preventing AIDS. The implementation of activities started being centrally managed by the MH, in a way to invert the relationship with the states, which by then created and executed their specific programsw.AIDS was included in Brazil’s 8th National Health Conference, within “Epidemiological Surveillance” sub-topic. Later, the MH coordinated debates on “AIDS and Constitutional Convention” topic in the states, and this subsidized the national 1987 campaign, which discussed how the new health care system could solve the AIDS issueThe analysis of documents and interviews showed that Lair Guerra’s administration was marked by NGO criticism related to the care of patients, to the campaigns, and to the official discourse, which was considered normative and medicalizing. Criticism was mainly given against 1988’s campaign, which held individuals accountable for measures they were not capable of taking, such as controlling their blood quality, in a way to shift responsibility from the state to the people. Nonetheless, there were also people supporting the government campaign .x. This issue was mentioned by some subjects interviewed, and it shows the importance of analyzing the roles of agents in this field in setting the policy.The relationship between the disease, sexuality, and use of injection drugs led its concept to be influenced by the religious discourse. Catholic tradition especially instilled the impression the disease was related to sin, moral transgression, and divine punishment, opposite to the discourse from homosexual groupsSíndrome da Imunodeficiência Adquirida – SIDA, in Portuguese), the foreign term was incorporated by physicians and the press in Brazil. Was the incorporation of a foreign word submission from scientific and cultural field agents to the USA, or simply a wish for better international communication in the scientific field? When asked, one of the interviewed subjects shows that, in the MH, its adoption was rationalized and formalized in a meeting for defining the term to be used.When it started being referred to as Acquired Immunodeficiency Syndrome (AIDS) .23 and in the lobby in the constitutional convention for pushing the blood issuey.The associations that fight AIDS (NGO/AIDS), which rose from 1985, had a determining role for regulating the control of blood and blood productsThe empirical material showed that the main disputes between agents in the PNAids and in the association movement revolved around the preventive campaigns . And from the release of azithromycin (AZT), also regarding treatment.During the whole period analyzed, the technical conception from the bureaucratic field prevailed in the official discourse. However, due to the influence from militant and religious fields, AIDS was considered to be a threat to society in general rather than restricted to specific groups.19.Lair’s international negotiations with the CDC and PAHO, as well as her management ability, contributed to the initial structuring and extension of PNAids. The Program also had important support from the Special Public Health Care Services Foundation for its interiorization, through its representative for transmittable diseases, sanitarian physician Pedro Chequer, who later was part of PNAids team19 was created. It was no longer part of DNDS and had autonomy regarding public health dermatology, a medical specialty. Its creation and transference to Brazil’s National Office of Basic Health Care Initiatives were made official in 1988. Lair remained in the management of the division until March 1990, during the early Collor administration.Getting PNAids attached to DNDS was under dispute, and this resulted from disagreements between agents from the medical field (Fabíola and Maria Leide) and AIDS Space, which was consolidating itself and seeking autonomy (Lair Guerra) (E10). In 1987, after Fabíola Nunes left SNPES, Brazil’s National STD/AIDS DivisionThe following people affected by the epidemic engaged in AIDS Space: HIV positive people, people who were more vulnerable to AIDS ; their parents and friends; professionals related to the epidemic, especially physicians and researchers. Those who enter this space for personal reasons, in general, joined the militant sub-space, had diverse educational backgrounds, and important cultural capital, with master’s or PhD’s degrees. In the bureaucratic and scientific sub-spaces, physicians predominated; the scientific field agents had graduate diplomas or were attending such courses .Despite the importance of the gay movement in planning the first responses to the epidemic, the NGO/AIDS were created seeking get detached from this movement  .20, through public health militants taking over positions in these institutions, such as Hésio Cordeiro and Reinaldo Guimarães.The first NGO/AIDS were funded by the State: Aids Support and Prevention Group (Gapa) by SES-SP , and Brazilian Interdisciplinary AIDS Association (ABIA) by the National Social Security Institute of Medical Care (E29) and by FinepIn this sub-space, the main issues were funding, structure, and liaison with the PNAids, living with HIV/AIDS, strategies for prevention and use of the technical formulation “men who have sex with men” (MSM). Disputes were also between the NGO/AIDS and groups in which AIDS was a cross-sectional topic, NGOs with more political perspectives (ABIA) and NGO focusing on care (Gapa)  .In the scientific field, young researchers and new PhD engaged themselves, seeking independent and original research lines. The main disputes were between generations or institutions. What was at stake was the recognition of the scientific authority or the position of state expert  .In the medical field, the disputes were between dermatologists, infectious disease specialists, tropical medicine, and infectious and parasitic diseases  .In the bureaucratic field, the disputes were between State Health Secretariats and PNAids  . Some agThe meeting of conceptions from the different sub-spaces on AIDS contributed to a broad construction of the problem and actions, based on the dignity of patients and human rights. CNAids, as the State commission that concentrated scientific and militant powers, transmuted into bureaucratic power, had an important role in planning the official discourse, which enabled negotiations between agents from different sub-spaces.Ação Popular (Popular Action) Party (E26) Most agents who had political capital to some extent stood out in the militant space, taking over dominant positions, such as the management of NGO/AIDS or other associative organizations . Others had taken part in the sanitary movement , in the gay rights movement , and in the fight against dictatorship, in clandestine parties such as Brazil’s Communist Party , and izations .This socio-historical analysis allowed explaining how the initial demand from militants to SES-SP took place, highlighting the role of the scientific field, from the start of the epidemic. It also recovered the initial formulation of the policy at the national level, which had rarely been described in the literature, identifying the main agents involved and showing the implementation of initiatives already in the last months of the military government. It also showed the importance of the participation of sanitarians who took over government positions at that time.AIDS Space was historically constituted as a space for fighting for the organization of the response to the epidemic and public health care intervention. The authority to talk about the meaning of the disease, the methods to prevent, and treat it were under dispute, as well as strategies for controlling the epidemic. Its structure involved agents from the medical field, from “Collective Health Space”, from the bureaucratic field, from the gay rights movement, and from the scientific field, influenced by political, religious, and juridical fields. Later, movements of people with hemophilia, people with thalassemia and sex workers, and NGO/AIDS, composing the militant sub-space.The government response had the level of government that implemented the first initiatives as a specific aspect. Traditionally, the MH had a national policy, which was followed and enforced by the states. The initiatives against AIDS were started in the states, when MH denied the need for intervention. The national policy was implemented since 1985. The conditions that enabled including AIDS in the political agenda were the following: the democratic transition process; sanitarians taking over management positions at the MH; the quick spread of the epidemic; the existence of research groups of infectious and parasitic diseases, involving pathologists who became immunologists; the advancements in the medical field regarding the disease, and the development stage of the clinical and laboratory research; the organization of AIDS programs by some states; and the pressure from social movements and the press.The comprehension of the historical reasons that allowed the initial planning of this policy and the analysis of the paths of agents and struggles common to AIDS Space and to the “Collective Health Space” empirically show the relationship between the policy and the sanitary reform movement and its assumptions. Its origin, during a time when the sanitary reform movement was rising and when there was no specific therapy, contributed to the initial prioritization of prevention. The dominance of the medical field and the constitution of the Brazilian Unified Health System (SUS) contributed to a connection between preventive measures and ensured treatment, a component that is necessary for implementing a policy that is based on integral care, which is internationally considered as an example.


Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed. Escherichia coli and Vibrio cholerae cells could enter a distinct state called the viable but non-culturable (VBNC) state , antigen 43 β-subunit, TolC, and OmpT, was found in cells exposed to nutrient-limited phosphate buffered saline (PBS), and the most drastic changes showing 106 proteins being modulated were found in those exposed to natural seawater and light  agar supplemented with 20 μl of 3% hydrogen peroxide (H2O2) was only able to grow on HI agar with one-tenth concentration of H2O2 or less . Due to the short half-life of mRNA, a positive signal indicates the presence of mRNA and thus, presence of viable cells that carry out transcription  was first described by Kogure et al. . It was p-iodonitrotetrazolium violet (INT) assay based on the activity of electron transport system ppGpp production resulted in faster VBNC state induction ppGpp level  . Knowing that OxyR regulates the expression of ahpC in several Gram-negative bacteria in response to elevated ROS levels  in Acinetobacter baumannii in the presence of organic peroxides , ATP synthase (oxidative phosphorylation) and a homolog of the Staphylococcus aureus elongation factor EF-Tu . The repression of these genes seems in agreement with the decrease in metabolic activity observed in VBNC cells. On the other hand, three proteins were over-expressed in VBNC cells: a putative catabolite regulator protein (CAA09491), a homolog of the Listeria innocua elongation factor EF-Ts, and a homolog of the Streptococcus pneumoniae fructose bisphosphate aldolase. In this study, the genetic pathways of E. faecalis underlying the VBNC response appear to be, in part, the same as those leading to the starvation response, as indicated by the presence of similar expression profiles for certain proteins like enoyl-ACP reductase (phospholipid biosynthesis). Nonetheless, significant differences were also observed in the protein profiles between starved cells and VBNC cells. A protein homologous to the mannose-specific phosphotransferase system (PTS) of Clostridium acetobutylicum was only up-regulated in starved cells, and an unidentified protein (30 kDa at pI 6) was only detected in VBNC cells. While the specific function of this unidentified protein is still unclear, a comparison of 2D gels obtained from different cell states suggests that it may be a modified form of enoyl-ACP reductase.One line of research involves detecting proteins present exclusively in cells in the VBNC state  against H2O2 and organic peroxide in V. parahaemolyticus in the context of the VBNC state and found that ahpC2 alone played a significant role in the induction and maintenance of the VBNC state at 4°C. While an earlier study found an enhanced quantity of AhpC2 protein in the VBNC cells   of non-culturable cells, which could not be recovered by supplementing with antioxidants, was resuscitated by another well-known resuscitation stimulus, an autoinducer (discussed in section Mechanisms of Resuscitation). These results suggest that P1 is composed of injured cells and P2 is composed of VBNC cells, so the increase in the number of culturable cells on antioxidant-supplemented agar was probably due to the recovery of injured cells instead of the resuscitation of VBNC cells.Interestingly, several studies have reported the unsuccessful resuscitation of VBNC cells after adding sodium pyruvate, catalase or superoxide dismutase onto agar plates  and autoinducers  , but the resuscitation of VBNC cells only became detectable an additional 2 h later  . It is also well established that once the selective pressures for latency is absent, mainly in the form of immunosuppression, these dormant bacteria can cause active tuberculosis (TB). The VBNC state of M. tuberculosis (Mtb) is also known as latency or dormancy, and contributes to over two million Mtb carriers around the world  and in vivo (Cornell model) models are used to generate VBNC cells of M. tuberculosis in order to study bacterial and host factors contributing to the VBNC status . Mtb-derived Rpfs have been shown to increase the culturability of VBNC bacteria in clinical sputum samples , but it is still used as a long-term therapy to treat the latent phase of disease , ethanol , high salinity (c. 290 ppt for 2 h), oxidative stress (0.2 mM H2O2 for 1 h), acidity (pH 3 for 25 min), antibiotics  and zinc (3.4 mM ZnSO47H2O for 1 h) .Because VBNC bacteria have a low metabolic rate, it is reasonable that antibacterials that target activities or components of active cells would be less effective against them. VBNC cells inside biofilms have an additional protection by the ECM. Moreover, the hypothesis that the VBNC state is an adaptation to stressful environments means that, by definition, these cells are likely to be less sensitive to exogenous stress. In M. tuberculosis infections clearly proves that VBNC cells in the latent phase of the disease are able to retain or, in the least, regain their virulence potential upon resuscitation. In fact, since the VBNC state is a slow metabolic state, it would seem more likely that the observed virulence is a result of reactivation of the normal function of the cell (Oliver, L. pneumophila has been demonstrated to retain the capacity to infect the amoeba used for resuscitation, demonstrating virulence toward its natural host (Steinert et al., L. pneumophila strain showed continued production of virulence-related proteins in VBNC cells but could not be resuscitated in the same amoeba (Alleron et al., Vibrio spp. have the ability to cause disease after resuscitation in their respective hosts (Baffone et al., Vibrio spp. revealed that a number of virulence and toxin genes were expressed in the VBNC state (Vora et al., S. typhi was also able to infect and cause disease in mouse models after resuscitation (Zeng et al., E. coli O157:H7 was shown to produce Shiga-like toxins, but the toxin levels produced were dependent upon the age and the inducing condition of the VBNC cells (Liu et al., The recurrence of  Oliver, . In suppP. aeruginosa is an important pathogen in terms of the disease burden and mortality associated with infection. It colonizes lung tissues of cystic fibrosis patients and is difficult to completely eradicate as it resides in biofilms in the lungs (Mulcahy et al., P. aeruginosa in this environment.These data on VBNC virulence suggests that, much like VBNC inducing stimuli and resuscitation factors, the expression and maintenance of virulence in different species of VBNC cells can vary greatly. M. tuberculosis, L. pneumophila, and P. aeruginosa but also in relation to food safety (Dinu and Bach, P. aeruginosa, great strides toward reducing the bacterial load in CF patients could be made by inducing resuscitation of the VBNC bacteria inside the human host which would then be more susceptible to antibiotics. It is an avenue of research that is worth exploring.This capacity of resuscitation with no apparent loss of virulence potential evidently brings about concerns regarding the presence of VBNC bacteria not only in a medical context with respiratory pathogens like After decades of study, it is clear that the VBNC state is both an important tool for the survival of bacteria and a dangerous aspect of bacterial pathogens for the host. The knowledge about the VBNC state comes from research on a variety of bacteria and highlights the complexity of this mechanism of adaptation. What seems clear is that induction and resuscitation of the VBNC state are highly variable across bacterial species and in some cases, strains. However, the basic genetic mechanisms may share a common theme and further research into this field will help tie up the loose ends that exist in this area. The ability to avoid conditions that lead to resuscitation, or the development of drugs that induce resuscitation during antibiotherapy could have a major impact on the consequence of the VBNC state in chronic infectious diseases. Development of new, inexpensive methods to easily detect cells in the VBNC state is needed to increase food safety. In conclusion, the potential applications of VBNC research are significant to prevent food- and water-borne infections, and find new treatments to cure chronic bacterial infections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Complicated intra-abdominal infections (cIAIs) are a common cause of morbidity worldwide, and in spite of improvements in patient care, therapeutic failure still occurs, impacting in-hospital resource consumption. This study aimed to assess the costs associated with the treatment of community-acquired cIAIs, from the Italian National Health Service perspective.This retrospective study analyzed the charts of patients who were discharged from four Italian university hospitals between January 1 and December 31, 2009 with a primary diagnosis of community-acquired cIAIs. Patient characteristics, diagnosis, surgical procedure, antibiotic therapy, and length of hospital stay were all recorded and the cost of total hospital care was estimated. Costs were calculated in Euros at 2009 values.The records of 260 patients  were analyzed. The average cost of care for a patient hospitalized due to cIAI was €4385 (95% CI 3650–5120), with an average daily cost of €419 (95% CI 378–440). Antibiotic therapy represented just under half (44.3%) of hospitalization costs. The strongest predictor of the increase in hospital costs was clinical failure: patients who clinically failed received an average of 8.2 additional days of antibiotic therapy and spent 11 more days in hospital compared with patients who responded to first-line therapy (both p < 0.05 vs. patients who were successfully treated). Furthermore, they incurred €5592 in additional hospitalization costs  with 53% (€2973) of the additional costs attributable to antibiotic therapy. Overall, antibiotic appropriateness rate was 78.8% (n = 205), and was significantly higher in patients receiving combination therapy compared with those treated with monotherapy (97.3% vs. 64.6%).The results of this study suggest that hospitals need to be aware of the clinical and economic consequences of antibiotic therapy of cIAIs and to reduce overall resource use and costs by improving the rate of success with appropriate initial empiric therapy. Intra-abdominal infections (IAIs), encompassing a wide spectrum of pathological conditions from uncomplicated appendicitis to fecal peritonitis, are a common cause of morbidity worldwide. IAIs are defined as complicated (cIAIs) when infection extends beyond the affected hollow viscus into the peritoneal space, causing either localized or diffuse peritonitis. In spit.The clinical outcome of patients with cIAIs is heavily influenced by prompt surgical source control and timely effective antimicrobial treatment,8. CurreMost recent guidelines also consider the antibiotic treatment of cIAIs from a microbiological point of view, particularly in terms of pathogens producing ESBLs (Extended Spectrum Beta-Lactamases). For community-acquired extrabiliary cIAIs, empirical antimicrobial therapy can be divided into categories: treatment for critically ill and non-critically ill patients, and treatment for both groups according to the presence or absence of risk factors for ESBL-producing pathogens. In non-critically ill patients, amoxicillin-clavulanate or ciprofloxacin-metronidazole are possible options, but in the presence of risk factors for ESBL these are not sufficient, and other drugs such as tigecycline and ertapenem are useful. In critically ill patients without risk factors for ESBL, piperacillin-tazobactam is an option, but in the presence of ESBL risk factors carbapenems like imipenem and meropenem are more appropriate.Of note, knowledge of antibiotic drugs costs is suggested as additional criteria supporting clinical decision-making,5,9. In To the best of our knowledge, a costs analysis of cIAIs hospital care has never been performed in Italy, although IAIs have been ranked as the second most common infectious reason for hospitalization, after respiratory infections. To addrThis one-year, multicentre, retrospective, incidence-based observational study was performed in four Italian  acute-care university hospitals. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki (and subsequent revisions) and to the current norm for observational studies. The protocol was reviewed and approved by each study site’s ethical committees. Due to the retrospective study design, informed consent was not deemed necessary.Patients were identified by searching computerized hospitalization records of each recruiting hospital for a primary discharge diagnoses of one of five International Classification of Diseases 9th revision (ICD-9) codes suggestive of cIAIs  were hospitalized between January 1 and December 31, 2009; (2) were at least 18 years old at the time of their hospitalization; (3) had a primary discharge diagnosis suggesting any cIAIs; (4) underwent laparotomy, laparoscopy or percutaneous drainage of an intra-abdominal abscess and (5) received intravenous antibiotics.A review of each patient’s chart was performed, and relevant parameters were recorded in standardized individual electronic case report forms. These included: patient age, gender, comorbidities , patient lifestyle factors , known risk factors for antibiotic failure,9  tract , gall-bladder, appendix, lower GI tract (colon-rectum), peritoneal abscesses drainage, or others.Clinical success was defined as patient recovery with either first line empiric antibiotic therapy or a step-down from initial therapy .Clinical failure was defined as a switch to second-line antibiotic treatment, need for unscheduled additional abdominal surgeries, or patient death[nt death-4,6,7.First-line empiric antibiotic therapy was defined as a regimen started at the time of surgical intervention, before the availability of any culture data.Switch to second-line antibiotic therapy was defined as the addition of one or more parenteral antibiotics to the initial antibiotic regimen or as a complete or partial switch of the initial antibiotic regimen to another parenteral antibiotic regimen.Unscheduled additional abdominal surgeries were taken into account if they occurred 2 or more days after the primary surgical procedure and were related to poor primary source control. Secondary procedures were not considered in the analysis when there was a mention of other reasons  that might have led to re-operation.appropriate if all isolated bacteria were sensitive to at least one of the antibiotics administered in patients with documented positive intra-abdominal swabs or blood cultures. Alternatively, in patients with negative or no cultures, empiric therapy was deemed as appropriate when the selected regimen covered enteric gram-negative aerobic and anaerobic bacteria and drug dosing was adequate, according to current guidelines[inappropriate.First-line empiric antibiotic therapy was defined as idelines. AntibioLeucocytosis was defined by a white blood cell (WBC) count >12,000/mm3. Leukopenia was defined as a WBC count <4000/mm3.A estimate of the cost of antibiotics was performed by multiplying the number of antibiotic days by the unit price of that antibiotic and by the number of per day doses. The overall cost of antibiotic treatment for each patient was the sum of costs calculated for all parenteral antibiotics received by the patient during the hospitalization period. The unit price of antibiotics was based on official ex-factory prices per unit in Italy. Laborata priori defined threshold , assuming that by subtracting the average costs of specialist services provided from this tariff, an acceptable proxy of the general cost sustained for patient management could be obtained.Other direct costs, including personnel, ordinary maintenance and hotel costs, were indirectly estimated by using Diagnosis-Related Group’s tariffs per admission provided to hospitals by the I-NHS. Specifically, this estimate was based on the acknowledged over-threshold per hospital day tariff, which is the per day cost to hospitals for length of stay prolonged over an Costs were expressed in Euro values at the time they were incurred .Sample size derived from the number of cIAIs patients meeting inclusion criteria, admitted at the four recruiting centers from January 1 to December 31, 2009.Continuous data are expressed as means ± standard deviation or 95% confidence intervals (CIs), and categorical data as number of events and percentages. Univariate statistical analysis was performed by student t-test or chi-squared test, as appropriate, to compare baseline characteristics and outcomes of clinical success and failure groups.Due to the retrospective design of the study, a regression model by means of a backward stepwise model selection approach was employed to investigate the independent hospital charges predictors, in order to control for confounding factors and obtain the exact contribution of each parameter to the outcome variable. The model takes into account patient status and controls for type of primary surgical procedure, unplanned additional surgeries, and antibiotic therapy switches. Considered variables were dummy. In order to avoid co-linearity between variables, a Pearson correlation was performed. Covariates in the model were: patient age and gender, one or more high risk factors, primary surgical procedure, surgical approach, antibiotic monotherapy/combination therapy, clinical success/failure, one or more therapeutic failure risk factors, unplanned additional surgeries, more than one additional surgery.P value <0.05 was considered statistically significant.Statistical analyses were performed by using SPSS statistical software version 15.1 . A A total of 260 patients  met the study entrance criteria. On hospital arrival, 250 (96.2%) patients were admitted to surgical wards, 8 3.1%) to medical wards, and 2 (0.7%) to the ICU. The majority of patients (62.3%) were affected by complicated appendicitis. Patients were surgically approached by laparoscopy in slightly more than half of cases, and by laparotomy in the majority of the others (Table % to mediEscherichia coli (58.8%), followed by Klebsiella pneumoniae (14.7%).Of the 48 microbiologically evaluable patients , 23 (47.9%) intra-operative abdominal site cultures , 12 (25.0%) abdominal drainage fluid cultures, 11 (22.9%) blood cultures and 2 (4.2%) surgical wound swabs were performed. Among 34 (70.8%) documented positive cultures, the most frequent isolated pathogen was Due to the low representation of the microbiological evaluable population, antibiotic therapy appropriateness was inferred by covered antimicrobial spectrum and dosing adequacy of starting empiric regimens, as detailed in the methods section. Overall, antibiotic appropriateness rate was 78.8% (n = 205), and was significantly higher in patients receiving combination therapy compared with those treated with monotherapy (97.3% vs. 64.6%). Clinical success chances with appropriate antibiotic therapy were 78.5% (n = 161) and 34.5% (n = 19) with inappropriate therapy.In total, 194 (74.6%) patients responded to antibiotic treatment and experienced clinical success , while 66 (25.4%) patients did not respond to antibiotic therapy . Ninety-six per cent (95.8%) of patients were discharged to home, 1.5% to long-term care facilities, 0.4% to another hospital, and 2.3% died in hospital.2 = 0.47) , with an average daily cost of €419 (95% CI 378–440). Antibiotic therapy cost by itself represented just under half (44.3%) of hospitalization costs. Clinical failure was the strongest independent predictor of hospitalization costs increases in multivariable regression analysis, followed by unscheduled additional abdominal surgeries, combination antibiotic therapy administration, patient comorbidities and illness severity markers  switched to second-line antibiotic therapy, 12 (18.2%) underwent unscheduled additional surgeries and 6 (9.1%) died. Second-line antibiotic therapy included switching to entirely different antibiotics in 63.6% of cases and addition of one or more drugs to the initial antibiotic regimen in 36.3% of cases. Reasons for switching therapy were clinical ineffectiveness in 63.6% of patients, microbiologic resistance in 9% and was unreported in 24.2% of patients. Second-line regimens involved meropenem (25.7%), ertapenem (21.2%), tygecicline (19.6%) and glycopeptides (10.6%).Patients who failed antibiotic therapy received an average of 8.2 additional days of antibiotic therapy and spent 11 more days in hospital compared with patients who responded to first-line therapy . Furthermore, they incurred €5592 in additional hospitalization costs  with 53% (€2973) of the additional costs attributable to antibiotic therapy  for patients experiencing clinical success, and €476 (95% CI 414–539) for patients who failed first-line therapy. Antibiotic cost by itself still was a great contributor to total per day inpatient charges, in both success and failure groups , being significantly higher in patients who failed starting therapy (€249 vs. €153).Due to the high contribution of antibiotic therapy to hospitalization costs, daily charges limited to antibiotic therapy course duration have been estimated (Figure To our knowledge, this is the first multicenter study investigating the economic outcome of hospitalized cIAIs in Italy. This study clearly shows that starting empirical antibiotic therapy has a large impact on the cost of care of community-acquired cIAIs. In this large sample of hospitalized adult patients with community-acquired cIAIs, clinical failure was the strongest independent predictor of increases in hospitalization costs. Compared with patients who are treated successfully, patients who failed therapy received antibiotic therapy for more than one additional week, spent 11 more days in hospital, and incurred a mean €5600 more in hospital charges. Antibiotic therapy was the leading contributor to inpatient charges, and multiple drug regimens was an independent predictor of increases in costs.Various European and US studies have investigated the clinical outcomes associated with the treatment of community-acquired cIAIs and have shown a clinical failure rate of 17%–35%-5, whichEarly European series have shown that hospitalization costs are 1.2–1.5 times higher in patients who have failed treatment compared with patients who were treated successfully. The preThe cost of antibiotic therapy explained over 50% of total hospitalization charges, confirming previous studies that have shown that antibiotics contribute 70% of extra costs associated with cIAIs. This laIn most cases, clinical failure with antibiotic therapy is driven by unsuitable drug choice,4,6. In As expected, the appropriateness of empiric antibiotic therapy was more frequently reached with wide spectrum combination therapy. We found that multiple-drug empiric regimens were appropriate in 97% of cases compared with roughly 65% of single drug regimens. Moreover, patients who achieved clinical success were more likely to have received antibiotic combination therapy than those patients who failed antibiotic therapy, confirming previous findings[Multiple antibiotic regimens aim to expand antimicrobial spectrum and to overcome increased bacterial resistance in community-acquired cIAIs,14. ReceAs previously reported,6,7, patThe low rate of intra-operative microbiology tests performed in the present study is worrisome. As choosing antibiotics for the treatment of cIAIs is an empiric decision, local epidemiology knowledge is of outmost importance. By increasing the chance of appropriate treatment, it coulAs with any retrospective analysis, this study has several limitations. Due to complexities associated with the collection of data, summary measures of illness and comorbidities severity, potentially associated with clinical failure, longer length of hospital stay, and higher inpatient costs were not covered and could not be used in the multivariate model. We were also unable to assess the appropriateness of antibiotic therapy in light of culture results and patient clinical risk profile,9 and, tDespite these limitations, for the first time we assessed patterns of starting antibiotic therapy, resource utilization and actual costs of caring for inpatients with community-acquired cIAIs in Italian hospitals. The results of this study suggest that hospitals need to be aware of the clinical and economic consequences of antibiotic therapy and to reduce overall resource use and costs by improving the rate of success with appropriate initial empiric therapy. Considering the prospective reimbursement system of the Italian NHS, there may be a relevant cost saving at the same reimbursement rate for hospitals, by reducing antibiotic costs of cIAIs. Mandatory peritoneal swab sampling, allowing for local epidemiology driven empiric antibiotic therapy, should be strongly encouraged for each cIAIs patient.CI: Confidence interval; cIAIs: Complicated intra-abdominal infections; GI: Gastrointestinal; IAIs: Intra-abdominal infections; ICU: Intensive care unit; WBC: White blood cell.The authors LD, FB, EC, FR and CA declare that they have no competing interests. SC has received funds from Pfizer. FS has received research and educational grants from Abbott, Bayer, Biogen Idec, Biomarine, BMS, Boehringer-Ingelheim, Celgene, Daiichi Sankyo, Eli Lilly, Genzyme, GlaxoSmithKline, Janssen Cilag, Johnson & Johnson, Medtronic, MSD, Novartis, Novo Nordisk, Obi, Pfizer, Roche, Sanofi Pasteur Servier, Sigma Tau, Stroder, Teva. AN has received funds to support the research and donations from Pfizer, MSD and honorary from Astra Zeneca, MSD, Pfizer, Valeas and Zambon.LD and CA carried out acquisition and interpretation of data, and drafted the manuscript; FB, SC, EC, AN, FR, and FS provided to conception and design of the study, and to manuscript revision; FS and CA performed the statistical analysis. All authors read and approved the final manuscript.


Immunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition. Forty-two cases with immunoglobulin G4-related sialadenitis (IgG4-RS) confirmed by histopathological and immunohistochemical assessment were studied to clarify the clinicopathologic characteristics of the salivary glands involved in IgG4-RS, especially the relationship between the histopathologic features and function of salivary glands or serum levels of IgG4.Clinical, serologic, imaging and histopathological data of these cases were analyzed. CT volumes of submandibular, parotid, and lacrimal glands were calculated. The saliva flow rate was measured. Scintigraphy with 99mTc-pertechnetate was undertaken in 31 cases, and the concentration index (CI) and secretion index (SI) was calculated. Relationships between fibrosis severity and salivary gland function or serum IgG4 levels were analyzed.The first symptom was swelling of bilateral submandibular or lacrimal glands. Physical examination showed multiple bilateral major salivary glands  and lacrimal glands were enlarged in IgG4 RS. Multiple enlarged cervical lymph nodes were noted in 30 patients. Saliva flow at rest was lower than normal in 34 cases; stimulated saliva flow was lower than normal in 15 cases. Secretory function was reduced more severely in the submandibular glands than in the parotid glands. Serum levels of IgG4 were elevated in 95.2% of cases and 78.6% patients had increased IgE levels. Serum IgG4 level was higher and saliva secretion lower as glandular fibrosis increased.Prominent changes in the morphology, histology, immunohistochemistry and secretion of the major salivary glands of IgG4-RS patients were accompanied by involvement of the lacrimal glands and cervical lymph nodes. Elevated IgE, allergic history, eosinophil infiltration suggest allergic reactions as a potential pathogenesis of IgG4-RS. Severity of glandular fibrosis correlated with salivary function and serum levels of IgG4. Immunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition. The disease is characterized by tumefactive lesions, a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, storiform fibrosis, and often  elevated serum concentrations of IgG4 in organs . IgG4-RDIgG4-RD has been described in virtually every organ system: the biliary tree, salivary glands, periorbital tissues, kidneys, lungs, lymph nodes, meninges, aorta, breast, prostate gland, thyroid gland, pericardium, and skin –6. SymptIdentification of IgG4-positive plasma cells in Küttner tumors and Mikulicz disease propelled renewed interest in these diseases, and fueled re-analyses of the classification of inflamed salivary glands –10. ConsSince IgG4-RS has been established as an entity in this century, clinicopathologic studies have been carried out in different countries , 11, 12.The present study was carried out to obtain more clinicopathologic information about IgG4-RS. In particular, we wished to clarify the characteristics of the salivary gland(s) involved in IgG4-RS. The relationship between the histopathologic features and function of salivary glands, the serum level of IgG4, and the number of IgG4-positive cells were analyzed to judge disease severity.The study protocol was approved by the Ethical Committee for Human Experiments of the Peking University School of Stomatology  and was conducted in accordance with the Declaration of Helsinki guidelines for human research. All patients provided informed consent prior to participation in this study.Forty-two patients were referred to the Department of Oral and Maxillofacial Surgery at Peking University School of Stomatology between August 2011 and June 2014. The patients were selected for this study because of bilateral swelling of submandibular glands with or without swelling of multiple exocrine glands  of >3 months duration. The diagnosis was made on the basis of symptoms, serologic analyses, imaging, and histopathological findings [Computed tomography (CT) was carried out with an eight-slice scanner . Volumes of the lacrimal, parotid, and submandibular glands were reconstructed by volume rendering , 16 and Whole saliva was collected as described previously , 19. Fiv99mTc-pertechnetate was undertaken in 31 cases using a standardized protocol [Scintigraphy with protocol . The conThe period from stimulation using 2.5 % citric acid solution to the minimum value after stimulation within 30 minutes was considered to be the “secretion phase”. The secretion index (SI) was calculated using the following formula:Mean values of bilateral sides were taken to be the CI and SI of the parotid and submandibular glands, respectively.Serologic analyses were undertaken for antinuclear antibody (ANA), IgE, IgA, IgM, total IgG, and subclasses of IgG using the Array 360 Immunoassay Assay Protein Serology Chemistry Analyzer system  by rate nephelometry immunoassay after generating charts and logs from quality control materials and the quality control. Anti-SS-A and anti-SS-B antibodies were tested by western blotting.2).All 42 cases underwent histopathologic examination. Hematoxylin and eosin-stained slides of all cases were reviewed by two pathologists (YC and YG) independently according to the diagnostic criteria for IgG4-RS , 14. ThrThe severity of inflammation and fibrosis was divided into three stages according to Seifert’s classification with slight modification . The firStage 1 referred to marked diffuse lymphoplasmacytic infiltration. Lymphoid follicles had developed. Fibrosis in the centers of lobules or periductal areas as well as atrophy of acini were noted Fig. . Stage 22O2. For antibodies against human IgG4 , IgG (Epitomics), and CD21 , antigen retrieval was achieved by treating sections with a boiling citric acid buffer solution  for 10 minutes in a microwave oven. Color was developed in freshly made diaminobenzidine. Sections were washed briefly in running tap water and stained lightly with Mayer’s hematoxylin. Negative controls were obtained by omission of the relevant primary antibody.Tissue sections (thickness 4 μm) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked using 3 % HThree identical HPFs with the greatest density of IgG-positive and IgG4-positive plasma cells were quantified. The ratio of IgG4-positive plasma cells to IgG-positive plasma cells was calculated for each patient.t test or the Wilcoxon rank sum test. P <0.05 was considered significant.Statistical analyses were performed using SPSS version 13.0 . Categorical variables were compared using the chi-square or Fisher exact test. Continuous variables are the mean ± standard deviation, and compared by the independent Of the 42 patients who comprised the study cohort, 16 were males and 26 were females with a ratio of 1:1.625. The median age was 55 (range 23–89) years.The first sign that patients found was bilateral swelling of the submandibular glands  or the lacrimal glands , followed by swelling of the unilateral submandibular gland , bilateral parotid gland , and bilateral sublingual gland . The interval between swelling of the first gland and swelling of the second gland was 1–96 (mean 22.6) months. Four (9.5 %) patients had AIP, six (14.3 %) cases had asthma, six (14.3 %) subjects had interstitial pneumonia, 22 (52.4 %) cases had rhinosinusitis, and two (4.8 %) cases had dysaudia.Physical examinations revealed that 64 of the total 69 submandibular glands  were enlarged in 27 bilateral cases and 10 unilateral cases. Twenty-two out of 83 parotid glands  were enlarged in nine bilateral cases and four unilateral cases. Fifty-five lacrimal glands were enlarged in 27 bilateral cases and one unilateral case. Bilateral enlargement of sublingual glands was observed in 22 patients  cases or from the parotid gland in five patients. The oral mucosa was wet with preservation of the saliva pool at the mouth floor in 28 patients. Mild dryness of the oral mucosa was found in seven (16.7 %) cases but the saliva pool had disappeared in 14 (33.3 %) cases. Rampant caries was not observed.Forty-one patients underwent CT examination. CT volumes of the enlarged submandibular, parotid and lacrimal glands are presented in Table The saliva flow rate at rest was 0.07–4.88 (mean 1.02 ± 0.94) g/5 minutes. Flow rates were lower than the normal value (1.9 g/5 minutes) in 34 (81.0 %) cases. The stimulated saliva flow rate was 2.06–17.34 (mean 8.76 ± 4.10) g/5 minutes, and nine (35.7 %) cases showed a lower stimulated saliva flow rate than the normal value (6.75 g/5 minutes) .99mTc-pertechnetate scintigraphy to evaluate the uptake and secretory function of submandibular and parotid glands. The mean CI of the submandibular glands was lower than normal value in 22.6 % patients, whereas the mean CI of the parotid glands was normal for all patients. The mean SI of the submandibular glands was lower than the normal value in 77.4 % patients, whereas the mean SI of the parotid glands was lower than the normal value in 45.2 % of subjects. Secretory function was reduced more severely in the submandibular glands than in the parotid glands.Thirty-one cases underwent In the 39 cases in whom the percentage of eosinophils in peripheral blood was measured, eight (20.5 %) cases showed elevated levels (7.7–14.1 %). The level of IgG was elevated in 53.8 % of patients. The level of IgG4 was elevated in 95.2 % of subjects. A total of 28.6 % of subjects had an elevated level of IgG2, and 78.6 % of patients had an increased level of IgE. The reaction to anti-SSA, anti-SSB, and anti-ANA was negative in all patients except for one patient who had a positive reaction to anti-SSA.Gross views revealed the involved glands to be enlarged and firm but their configurations to be maintained. The cut surface varied in different areas. In the most serious areas, gland tissue was homogeneous and appeared as an ill-defined mass. The color was white or pale yellow. Upon more careful study, the structure of glandular lobules could be ascertained. Uninvolved areas retained the appearance of a normal gland.Histological sections revealed preservation of the lobular architecture, marked lymphoplasmacytic inflammation, large irregular lymphoid follicles with expanded germinal centers, and acinar atrophy without prominent lymphoepithelial lesions. Characteristically, prominent cellular interlobular fibrosis was shown to be due to activated fibroblasts, and infiltration of lymphocytes and plasma cells was shown. Plasma cells were mature without sign of atypia. Eosinophil infiltration in the involved areas was noted in 20 cases (47.6 %), and the number ranged from 2/HPF to 98/HPF (median 16/HPF). Obliterative phlebitis was noted in 30 cases.According to our modified Seifert classification, out of 42 cases 14 patients were classified as stage-1 disease, stage-2 disease, and stage-3 disease, respectively.Numbers of IgG4-positive-stained plasma cells were increased in all cases. Mean numbers of IgG-positive and IgG4-positive plasma cells per HPF were 167 ± 42 (range 79–246) and 124 ± 42 (range 56–215), respectively. The IgG4/IgG ratio was 73.7 ± 12.8 % (range 50.4–93.9 %).P <0.05) as glandular fibrosis became more severe. Also, the stimulated flow rate and CI of the submandibular gland decreased significantly (P <0.05) as glandular fibrosis became more severe.Clinical features, serologic and immunohistochemical examinations, and secretion function of the salivary glands of patients with stage-1, stage-2 and stage-3 diseases are summarized in Table IgG4-RD is accepted as an immune-mediated disease involving multiple disorders such as sclerosing pancreatitis, sclerosing cholangitis, tubulointerstitial nephritis, interstitial pneumonia, retroperitoneal fibrosis, inflammatory pseudotumor of the liver and lung, inflammatory aortic aneurysms, and enlargement of the salivary and lacrimal glands , 23–25. Histopathological studies based on biopsy material involving IgG4-RS in the major salivary glands are few. Most histopathological information is derived from whole-gland ectomy specimens. We believe it is important to take biopsies from the involved major salivary gland  to make a definite diagnosis of IgG4-RS for two main reasons. First, a relatively large dose of glucocorticoids (“assault therapy”) in the short term and a small dose as maintenance therapy in the long term are typical first-line therapies . The resThe relatively superficial location of the submandibular glands enables incisional biopsy, which is difficult for affected internal organs such as the pancreas and kidneys. The biopsy was undertaken under local anesthesia. The superficial part of the glands was chosen for biopsy. Gland tissue of dimension 0.7 cm × 0.5 cm was excised for biopsy. The wound in the glands was closed carefully. No complications were found and secretory function was not affected.Usually, IgG4-RS involves swelling of multiple exocrine glands . This was true of our series. Gland volume was measured by CT volume rendering and was compared with normal values. Compared with the results of palpation, the number of enlarged parotid glands increased obviously according to the measurement of gland volume by CT. Twenty-two out of 83 parotid glands were enlarged in nine bilateral cases and four unilateral cases according to physical examination. Fifty-three of 81 (one patient did not undergo CT examination) parotid glands were enlarged in 24 bilateral cases and five unilateral cases according to measurement of the gland volume by CT. The main reason for these discrepancies between the two methods of measurement is that the anatomy of the parotid glands is irregular and difficult for accurate evaluation by palpation if the swelling is not obvious, whereas CT volume rendering can detect enlargement of the parotid glands accurately and quantitatively. The high prevalence of enlargement of the parotid glands in IgG4-RS should be noted, and CT volume rendering is advocated. Bilateral enlargement of the sublingual glands and accessory parotid glands is very rare in other diseases but is relatively common in IgG4-RS. In the present study, unexpected bilateral swelling of the sublingual glands (52.4 %) and accessory parotid glands (22.0 %) was observed. Bilateral swelling of the sublingual glands and/or accessory parotid glands is therefore an important clinical feature in the diagnosis of IgG4-RS.We agree with Geyer and Deshpande  that uniAs a component of IgG4-related systemic disease, careful monitoring of the pancreas, gallbladder, kidneys, and lungs should be undertaken to identify other systemic diseases in the diagnosis of IgG4-RS. The nose, paranasal sinuses, and ears are also involved very often along with the salivary and lacrimal glands in IgG4-RS. A total of 71.4 % cases in the present study had lymphadenopathy of the upper cervical lymph nodes. Swelling of the cervical lymph nodes was the first symptom and sign in three cases. Lymphadenopathy of the cervical lymph nodes should therefore also be considered an important clinical feature in the diagnosis of IgG4-RS.99mTc-pertechnetate are effective for evaluation of the function of the major salivary glands. These procedures are relatively easy to carry out, noninvasive, and well tolerated. Studies have shown that IgG4-RS leads to a mild reduction in saliva flow [99mTc-pertechnetate scintigraphy showed that the percentage of patients with a mean SI lower than the normal value was higher in the submandibular glands (77.4 %) than in the parotid glands (45.2 %), suggesting that damage to the submandibular glands was more severe than that to the parotid glands in IgG4-RS.Saliva flow rates and scintigraphy with iva flow . In the There is evidence of autoimmune disorder in AIP but there are also signs of an allergic nature in its pathogenesis. In our series, an elevated serum level of IgG4 was detected in 95.2 % of cases along with an obviously elevated level of IgE in 78.6 %. In one study of 42 patients with AIP, the IgG4 level was elevated in 93 % of cases and the IgE level was elevated in 86 % of cases . High leElevated IgE levels and eosinophil infiltration are among the criteria for the diagnosis of IgG4-RD . Serum cSeifert  put forwAs the severity of glandular fibrosis increased, serum levels of IgG and IgG4 in the three classes increased significantly, whereas the (IgG1 + IgG3) / (IgG2 + IgG4) ratio decreased significantly. These data suggest that the results of serological examinations have a good correlation with histopathologic grading, thereby enabling serological results to be used to estimate lesion severity.The stimulated flow rate of whole saliva decreased with increasing histopathological grade. Increased fibrosis was accompanied by a decreased CI in scintigrams in the submandibular glands, suggesting that the increase in inflammatory fibrosis and glandular function had declined. Treatment of IgG4-RS should therefore be initiated as early as possible once the definitive diagnosis is established to preserve the function of involved salivary glands.Biopsy is very important to provide the necessary information for the definitive diagnosis of IgG4-RS. Bilateral swelling of the sublingual glands and accessory parotid glands, lymphadenopathy of the cervical lymph nodes, and bilateral enlargement of parotid and submandibular glands are important clinical features of IgG4-RS. Elevated serum levels of IgE, allergic history, and eosinophil infiltration suggest allergic reactions as a potential pathogenesis. Increased fibrosis accompanied with lower salivary function suggests that the early diagnosis and treatment of IgG4-RS is very important.