Antibiotic Consumption, Illness, and Maternal Sensitivity in Infants with a Disorganized Attachment.

Prior research found an association between mother-infant attachment and antibiotic use. Ambivalent-attached infants are more likely to take antibiotics than other infants, and their mothers tend to be less sensitive to their needs than most. This finding is important because it shows the association between psychological processes, early relationships, and health outcomes. We aim to learn about children with high-risk attachment relationships, such as disorganized-attached infants. This study compares antibiotic use, infant-mother interactive behavior, and health indicators according to infant attachment patterns (including disorganized attachment). For this purpose, we observed mothers-infants' interactive behavior in free play at nine months and infants' attachment in the Ainsworth Strange Situation at twelve months. Participants included 77 girls and 104 boys (full-term and preterm) and their mothers. Paradoxically, mothers of disorganized-attached infants reported that their children were ill only 1.56 times on average, but 61% of their children used antibiotics in the first nine months. The other mothers reported that their children were sick 5.73 times on average, but only 54% of their children used antibiotics in the same period. Infants with disorganized attachment had mothers who were more literate and less sensitive. These results add to a body of research that shows that early high-risk relationships affect children's lives at multiple levels.

Multi-Drug Resistance in Bacterial Genomes-A Comprehensive Bioinformatic Analysis.

Antimicrobial resistance is presently one of the greatest threats to public health. The excessive and indiscriminate use of antibiotics imposes a continuous selective pressure that triggers the emergence of multi-drug resistance. We performed a large-scale analysis of closed bacterial genomes to identify multi-drug resistance considering the ResFinder antimicrobial classes. We found that more than 95% of the genomes harbor genes associated with resistance to disinfectants, glycopeptides, macrolides, and tetracyclines. On average, each genome encodes resistance to more than nine different classes of antimicrobial drugs. We found higher-than-expected co-occurrences of resistance genes in both plasmids and chromosomes for several classes of antibiotic resistance, including classes categorized as critical according to the World Health Organization (WHO). As a result of antibiotic-resistant priority pathogens, higher-than-expected co-occurrences appear in plasmids, increasing the potential for resistance dissemination. For the first time, co-occurrences of antibiotic resistance have been investigated for priority pathogens as defined by the WHO. For critically important pathogens, co-occurrences appear in plasmids, not in chromosomes, suggesting that the resistances may be epidemic and probably recent. These results hint at the need for new approaches to treating infections caused by critically important bacteria.

Plasmids Increase the Competitive Ability of Plasmid-Bearing Cells Even When Transconjugants Are Poor Donors, as Shown by Computer Simulations.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

Plasmid Costs Explain Plasmid Maintenance, Irrespective of the Nature of Compensatory Mutations.

Conjugative plasmids often carry virulence and antibiotic-resistant genes. Therefore, understanding the behavior of these extra-chromosomal DNA elements gives insights into their spread. Bacteria frequently replicate slower after plasmids' entry, an observation inconsistent with the plasmids' ubiquity in nature. Several hypotheses explain the maintenance of plasmids among bacterial communities. However, the numerous combinations of bacterial species and strains, plasmids, and environments claim a robust elucidatory mechanism of plasmid maintenance. Previous works have shown that donor cells already adapted to the plasmid may use the plasmid as a 'weapon' to compete with non-adapted plasmid-free cells. Computer simulations corroborated this hypothesis with a wide range of parameters. Here we show that donor cells benefit from harboring conjugative plasmids even if compensatory mutations in transconjugant cells occur in the plasmid, not on chromosomes. The advantage's leading causes are as follows: mutations take time to appear, many plasmids remain costly, and re-transfer of mutated plasmids usually occurs in sites distant to the original donors, implying little competition between these cells. Research in previous decades cautioned against uncritical acceptance of the hypothesis that resistance cost helps to preserve antibiotics' effectiveness. This work gives a new twist to this conclusion by showing that costs help antibiotic-resistant bacteria to compete with plasmid-free cells even if compensatory mutations appear in plasmids.

The Association between Prematurity, Antibiotic Consumption, and Mother-Infant Attachment in the First Year of Life.

Antibiotics have individual and public-health drawbacks. Nevertheless, mother-infant attachment quality and maternal sensitivity are associated with antibiotic use. Ambivalent-attached infants are more likely to consume antibiotics than other infants. Conceivably, the emotional over-externalization of ambivalent-attached infants and maternal anxiety when infants are ill raise concerns in healthcare professionals, leading to antibiotic over-prescriptions. However, because infants prematurely born, particularly those with less than 32 weeks of gestation, are under more accurate health vigilance, the impact of infant and maternal behavior on antibiotic prescription may vanish in this sample. To test this hypothesis, we performed a longitudinal study to compare antibiotic use and the quality of mother-infant attachment in three groups: 86 infants born at full-term, 44 moderate-to-late preterm infants (32-36 gestation weeks), and 58 very-to-extreme preterm infants (<32 gestation weeks). Infants' attachment was observed with the Ainsworth Strange Situation's experimental paradigm at 12 months of corrected age. Findings indicate that infant attachment strategy is associated with antibiotics uptake, but results vary across samples. The proportion of infants that used antibiotics is highest among ambivalent-attached infants in the full-term sample but highest among avoidant-attached infants in the very-to-extreme premature sample. Moreover, higher infant gestational age and lower maternal sensitivity determine higher antibiotic use.

The Impact of Non-Pathogenic Bacteria on the Spread of Virulence and Resistance Genes.

This review discusses the fate of antimicrobial resistance and virulence genes frequently present among microbiomes. A central concept in epidemiology is the mean number of hosts colonized by one infected host in a population of susceptible hosts: R0. It characterizes the disease's epidemic potential because the pathogen continues its propagation through susceptible hosts if it is above one. R0 is proportional to the average duration of infections, but non-pathogenic microorganisms do not cause host death, and hosts do not need to be rid of them. Therefore, commensal bacteria may colonize hosts for prolonged periods, including those harboring drug resistance or even a few virulence genes. Thus, their R0 is likely to be (much) greater than one, with peculiar consequences for the spread of virulence and resistance genes. For example, computer models that simulate the spread of these genes have shown that their diversities should correlate positively throughout microbiomes. Bioinformatics analysis with real data corroborates this expectation. Those simulations also anticipate that, contrary to the common wisdom, human's microbiomes with a higher diversity of both gene types are the ones that took antibiotics longer ago rather than recently. Here, we discuss the mechanisms and robustness behind these predictions and other public health consequences.

Psychological and cultural factors influencing antibiotic prescription.

Humans have inundated the environment worldwide with antimicrobials for about one century, giving selective advantage to antibiotic-resistant bacteria. Therefore, antibiotic resistance has become a public health problem responsible for increased mortality and extended hospital stays because the efficacy of antibiotics has diminished. Hospitals and other clinical settings have implemented stewardship measures to reduce antibiotic administration and prescription. However, these measures demand multifactorial approaches, including multidisciplinary teams in clinical settings and the education of professionals and patients. Recent studies indicate that individual factors, such as mother-infant attachment and parenting styles, play a critical role in antibiotic use. Also, macrocontextual factors, such as economic, social, or cultural backgrounds, may impact antibiotic use rates. Therefore, research aiming to ameliorate stewardship measures must include psychologically and sociologically based research.

Estuarine Aquacultures at the Crossroads of Animal Production and Antibacterial Resistance: A Metagenomic Approach to the Resistome.

It is recognized that the spread of antibiotic resistance (AR) genes among aquatic environments, including aquaculture and the human environment, can have detrimental effects on human and animal health and the ecosystem. Thus, when transmitted to the human microbiome or pathogens, resistance genes risk human health by compromising the eventual treatment of infections with antibiotic therapy. This study aimed to define the resistance profile of aquaculture farms and their potential risk for spreading. Twenty-four sediments from oyster and gilthead sea bream aquaculture farms located in three Portuguese river estuaries (17 sediments from Sado, 4 from Aveiro, and 3 from Lima) were studied by comparative metagenomic analysis. The computation of the diversity of genes conferring resistance per antibiotic class revealed a significant increase in aminoglycosides, beta-lactams, disinfectants, quinolones, and tetracyclines counts. In all geographic locations under study, the most diverse AR genes confer resistance to the macrolides, tetracyclines and oxazolidinones classes, all of which are medically important for human and animal therapies, as well as resistance to disinfectants. The diversity of mobile genetic elements correlated with the number of AR genes such as tetracyclines, suggesting that AR could be easily mobilized among bacterial genomes and microbiomes.

The impact of low birthweight in infant patterns of regulatory behavior, mother-infant quality of interaction, and attachment.

It remains unclear whether infants born preterm are more likely to develop an insecure attachment with their mothers. In this study, instead of using gestational age criteria, we observe attachment in infants born with very low birthweight. Although the collinearity between gestational age and birthweight is high, infants born with very low birthweight for their gestational age tend to stay more days in NICU and to have more comorbidities than other infants with the same gestational age. Thus, we wonder about the impact of low gestational birth (per se) in infants' regulatory behavior, the quality of mother-infant interactions, and attachment security. The participants are 71 infants' weight lower than 1599 g of gestational weight (varying between 23 and 34 weeks of gestational) and their mothers. Dyads were observed in free play and during Face to Face Still-Face paradigm with infants at 3 months of corrected age. At 12 months of corrected age, mother-infant attachment was observed during Strange Situation. Results indicate that infants with low/very low gestational birthweight have high levels of insecure attachment (70 %) and non-positive patterns of regulatory behavior (64 %). Maternal and infant interactive behavior is highly associated with infant attachment. In turn, maternal interactive behavior is associated with gestational age, birthweight, and number of days in NICU.

Are Virulence and Antibiotic Resistance Genes Linked? A Comprehensive Analysis of Bacterial Chromosomes and Plasmids.

Although pathogenic bacteria are the targets of antibiotics, these drugs also affect hundreds of commensal or mutualistic species. Moreover, the use of antibiotics is not only restricted to the treatment of infections but is also largely applied in agriculture and in prophylaxis. During this work, we tested the hypothesis that there is a correlation between the number and the genomic location of antibiotic resistance (AR) genes and virulence factor (VF) genes. We performed a comprehensive study of 16,632 reference bacterial genomes in which we identified and counted all orthologues of AR and VF genes in each of the locations: chromosomes, plasmids, or in both locations of the same genome. We found that, on a global scale, no correlation emerges. However, some categories of AR and VF genes co-occur preferentially, and in the mobilome, which supports the hypothesis that some bacterial pathogens are under selective pressure to be resistant to specific antibiotics, a fact that can jeopardize antimicrobial therapy for some human-threatening diseases.

Maternal sensitivity and mother-infant attachment are associated with antibiotic uptake in infancy.

Attachment security has been associated with health status and symptom reporting. In this longitudinal study, we investigated the association between antibiotics uptake by infants at 9-months and mother-infant attachment at 12-months. Logistic regression analyses indicated that lower maternal sensitivity was associated with increased odds of antibiotic uptake. Furthermore, 89.7% of insecure-ambivalent infants consumed antibiotics, which contrasted with 32.5% of avoidant infants and 21.5% of secure infants. This study suggests that maternal behavior and mother-infant attachment impact on antibiotic consumption, which is worrying because antibiotics may lead to several health problems later in life and antibiotic-resistance.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids.

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a 'weapon' to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Bacterial persistence is essential for susceptible cell survival in indirect resistance, mainly for lower cell densities.

Antibiotic-susceptible bacteria may survive bactericidal antibiotics if other co-inhabiting bacteria detoxify the medium through antibiotic degradation or modification, a phenomenon denominated as indirect resistance. However, it is unclear how susceptible cells survive while the medium is still toxic. One explanation relies on the speed of detoxification, and another, non-exclusive explanation, relies on persistence, a state of bacterial dormancy where cells with low metabolic activity and growth rates are phenotypically tolerant to antibiotics and other cytotoxic substances. Here we simulated the fate of susceptible cells in laboratory experiments in the context of indirect resistance to understand whether persistence is necessary to explain the survival of susceptible cells. Depending on the strain and experimental conditions, the decay of persister populations may follow an exponential or a power-law distribution. Therefore, we studied the impact of both distributions in the simulations. Moreover, we studied the impact of considering that persister cells have a mechanism to sense the presence of a toxic substance-a mechanism that would enable cells to leave the dormant state when the medium becomes nontoxic. The simulations show that surviving susceptible cells under indirect resistance may originate both from persister and non-persister populations if the density of detoxifying cells is high. However, persistence was necessary when the initial density of detoxifying cells was low, although persister cells remained in that dormancy state for just a few hours. Finally, the results of our simulations are consistent both with exponential and power-law decay of the persistence population. Whether indirect resistance involves persistence should impact antibiotic treatments.

COVID-19 Lockdowns May Reduce Resistance Genes Diversity in the Human Microbiome and the Need for Antibiotics.

Recently, much attention has been paid to the COVID-19 pandemic. Yet bacterial resistance to antibiotics remains a serious and unresolved public health problem that kills hundreds of thousands of people annually, being an insidious and silent pandemic. To contain the spreading of the SARS-CoV-2 virus, populations confined and tightened hygiene measures. We performed this study with computer simulations and by using mobility data of mobile phones from Google in the region of Lisbon, Portugal, comprising 3.7 million people during two different lockdown periods, scenarios of 40 and 60% mobility reduction. In the simulations, we assumed that the network of physical contact between people is that of a small world and computed the antibiotic resistance in human microbiomes after 180 days in the simulation. Our simulations show that reducing human contacts drives a reduction in the diversity of antibiotic resistance genes in human microbiomes. Kruskal-Wallis and Dunn's pairwise tests show very strong evidence (p < 0.000, adjusted using the Bonferroni correction) of a difference between the four confinement regimes. The proportion of variability in the ranked dependent variable accounted for by the confinement variable was η2 = 0.148, indicating a large effect of confinement on the diversity of antibiotic resistance. We have shown that confinement and hygienic measures, in addition to reducing the spread of pathogenic bacteria in a human network, also reduce resistance and the need to use antibiotics.

The Perfect Condition for the Rising of Superbugs: Person-to-Person Contact and Antibiotic Use Are the Key Factors Responsible for the Positive Correlation between Antibiotic Resistance Gene Diversity and Virulence Gene Diversity in Human Metagenomes.

Human metagenomes with a high diversity of virulence genes tend to have a high diversity of antibiotic-resistance genes and vice-versa. To understand this positive correlation, we simulated the transfer of these genes and bacterial pathogens in a community of interacting people that take antibiotics when infected by pathogens. Simulations show that people with higher diversity of virulence and resistance genes took antibiotics long ago, not recently. On the other extreme, we find people with low diversity of both gene types because they took antibiotics recently-while antibiotics select specific resistance genes, they also decrease gene diversity by eliminating bacteria. In general, the diversity of virulence and resistance genes becomes positively correlated whenever the transmission probability between people is higher than the probability of losing resistance genes. The positive correlation holds even under changes of several variables, such as the relative or total diversity of virulence and resistance genes, the contamination probability between individuals, the loss rate of resistance genes, or the social network type. Because the loss rate of resistance genes may be shallow, we conclude that the transmission between people and antibiotic usage are the leading causes for the positive correlation between virulence and antibiotic-resistance genes.

Plasmid Interactions Can Improve Plasmid Persistence in Bacterial Populations.

It is difficult to understand plasmid maintenance in the absence of selection and theoretical models predict the conditions for plasmid persistence to be limited. Plasmid-associated fitness costs decrease bacterial competitivity, while imperfect partition allows the emergence of plasmid-free cells during cell division. Although plasmid conjugative transfer allows mobility into plasmid-free cells, the rate of such events is generally not high enough to ensure plasmid persistence. Experimental data suggest several factors that may expand the conditions favorable for plasmid maintenance, such as compensatory mutations and accessory genes that allow positive selection. Most of the previous studies focus on bacteria that carry a single plasmid. However, there is increasing evidence that multiple plasmids inhabit the same bacterial population and that interactions between them affect their transmission and persistence. Here, we adapt previous mathematical models to include multiple plasmids and perform computer simulations to study how interactions among them affect plasmid maintenance. We tested the contribution of different plasmid interaction parameters that impact three biological features: host fitness, conjugative transfer and plasmid loss - which affect plasmid persistence. The interaction affecting conjugation was studied in the contexts of intracellular and intercellular interactions, i.e., the plasmids interact when present in the same cell or when in different cells, respectively. First, we tested the effect of each type of interaction alone and concluded that only interactions affecting fitness (epistasis) prevented plasmid extinction. Although not allowing plasmid maintenance, intracellular interactions increasing conjugative efficiencies had a more determinant impact in delaying extinction than the remaining parameters. Then, we allowed multiple interactions between plasmids and concluded that, in a few cases, a combined effect of (intracellular) interactions increasing conjugation and fitness lead to plasmid maintenance. Our results show a hierarchy among these interaction parameters. Those affecting fitness favor plasmid persistence more than those affecting conjugative transfer and lastly plasmid loss. These results suggest that interactions between different plasmids can favor their persistence in bacterial communities.

Dominance Between Plasmids Determines the Extent of Biofilm Formation.

Bacterial biofilms have an impact in medical and industrial environments because they often confer protection to bacteria against harmful agents, and constitute a source from which microorganisms can disperse. Conjugative plasmids can enhance bacterial ability to form biofilms because conjugative pili act as adhesion factors. However, plasmids may interact with each other, either facilitating or inhibiting plasmid transfer. Accordingly, we asked whether effects on plasmid transfer also impacts biofilm formation. We measured biofilm formation of Escherichia coli cells harboring two plasmid types, or when the two plasmids were present in the same population but carried in different cells. Using eleven natural isolated conjugative plasmids, we confirmed that some indeed promote biofilm formation and, importantly, that this ability is correlated with conjugative efficiency. Further we studied the effect of plasmid pairs on biofilm formation. We observed increased biofilm formation in approximately half of the combinations when both plasmids inhabited the same cell or when the plasmids were carried in different cells. Moreover, in approximately half of the combinations, independent of the co-inhabitation conditions, one of the plasmids alone determined the extent of biofilm formation - thus having a dominant effect over the other plasmid. The molecular mechanisms responsible for these interactions were not evaluated here and future research is required to elucidate them.

The Social Distancing Imposed To Contain COVID-19 Can Affect Our Microbiome: a Double-Edged Sword in Human Health.

Hygienic measures imposed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and contain COVID-19 have proven effective in controlling the pandemic. In this article, we argue that these measures could impact the human microbiome in two different and disparate ways, acting as a double-edged sword in human health. New lines of research have shown that the diversity of human intestinal and oropharyngeal microbiomes can shape pulmonary viral infection progression. Here, we suggest that the disruption in microbial sharing, as it is associated with dysbiosis (loss of bacterial diversity associated with an imbalance of the microbiota with deleterious consequences for the host), may worsen the prognosis of COVID-19 disease. In addition, social detachment can also decrease the rate of transmission of antibiotic-resistant bacteria. Therefore, it seems crucial to perform new studies combining the pandemic control of COVID-19 with the diversity of the human microbiome.

Antibiotic Resistance Gene Diversity and Virulence Gene Diversity Are Correlated in Human Gut and Environmental Microbiomes.

Human beings have used large amounts of antibiotics, not only in medical contexts but also, for example, as growth factors in agriculture and livestock, resulting in the contamination of the environment. Even when pathogenic bacteria are the targets of antibiotics, hundreds of nonpathogenic bacterial species are affected as well. Therefore, both pathogenic and nonpathogenic bacteria have gradually become resistant to antibiotics. We tested whether there is still cooccurrence of resistance and virulence determinants. We performed a comparative study of environmental and human gut metagenomes from different individuals and from distinct human populations across the world. We found a great diversity of antibiotic resistance determinants (AR diversity [ARd]) and virulence factors (VF diversity [VFd]) in metagenomes. Importantly there is a correlation between ARd and VFd, even after correcting for protein family richness. In the human gut, there are less ARd and VFd than in more diversified environments, and yet correlations between the ARd and VFd are stronger. They can vary from very high in Malawi, where antibiotic consumption is unattended, to nonexistent in the uncontacted Amerindian population. We conclude that there is cooccurrence of resistance and virulence determinants in human gut microbiomes, suggesting a possible coselective mechanism.IMPORTANCE Every year, thousands of tons of antibiotics are used, not only in human and animal health but also as growth promoters in livestock. Consequently, during the last 75 years, antibiotic-resistant bacterial strains have been selected in human and environmental microbial communities. This implies that, even when pathogenic bacteria are the targets of antibiotics, hundreds of nonpathogenic bacterial species are also affected. Here, we performed a comparative study of environmental and human gut microbial communities issuing from different individuals and from distinct human populations across the world. We found that antibiotic resistance and pathogenicity are correlated and speculate that, by selecting for resistant bacteria, we may be selecting for more virulent strains as a side effect of antimicrobial therapy.