Review

Are antibiotics a contributory factor to the rise in allergic and other Chronic Inflammatory Diseases?

An assessment of the evidence

Sally F Bloomfield, London School of Hygiene and Tropical Medicine, International Scientific Forum on Home Hygiene

October 2014

Summary

Increasingly there is evidence to suggest that antibiotic usage is a contributory factor in the rapid rise in allergy and other Chronic Inflammatory Diseases which has occurred since the 1970s. Since 1999 epidemiological and other studies have shown that antibiotics, particularly excessive antibiotic use during pregnancy or the neonatal period, induce changes in the microbiota of the human body, and that these changes are implicated in the development of asthma, eczema, cow’s milk allergy and irritable bowel disease (IBD).

A key strategy being developed to address the global problem of antibiotic resistance is “antibiotic stewardship” - the control and reduction of antibiotic prescribing. If antibiotic overprescribing is a causative factor in inflammatory diseases as well as in the development of antibiotic resistance, this provides a further powerful argument which could also be used to discourage the public from seeking antibiotic treatments for their children, for minor infections or infections which are viral in origin.

This IFH report reviews the growing numbers (more than 40) of epidemiological studies which have evaluated the possible link between antibiotic usage and the development, primarily of asthma, but also eczema, cow’s milk allergy and irritable bowel disease (IBD). Although, there is still much concern and discussion that there may have been some overestimation of the association, due to confounding factors such as reverse causation, many or most workers now agree that there is strong evidence that antibiotic usage use, during pregnancy or the neonatal period, is a contributory factor in the development of these diseases, although the extent of this association is not clear, and certainly other factors are also involved.

The proposed explanation for this link is that broad spectrum antibiotics alter the gut microbiota, which in turn affects the maturing immune system in a way that promotes inflammatory disease development. This concept has its origins in the so-called “hygiene hypothesis”. The two more recent, and now more widely accepted, theories about this concept are that the human body requires exposure to a broad range of microbes,

particularly during early development, but that these are the largely non harmful microbes which make up the microbiota of our human (gut, skin etc), animal and natural environments.

Because of the difficulties associated with interpreting epidemiological studies, which stem from the large number of interrelated factors known to influence development of inflammatory diseases, animal studies are being increasingly used to better understand the relationship between antibiotic use and development of allergic disease. These studies, also reviewed here, not only support the concept of a link between antibiotics and allergies, but are beginning to reveal the mechanisms by which this can occur. Importantly they demonstrate that it is the altered microbiota which is associated with the increased risk of inflammatory diseases, not the antibiotic per se.

Although epidemiological studies largely support the concept of a link between antibiotics and allergic diseases, epidemiological studies exploring the link between antibiotics and sensitization/atopy have mainly failed to find an association. This suggests that other biological mechanisms may also be involved, particularly in case of asthma.

Introduction

The hygiene hypothesis dates from 1989 when Strachan proposed that lower incidence of infection in early childhood could be an explanation for the rapid 20th century rise in allergic diseases. Researchers now recognise that, whilst the concept of a link between microbial exposure and allergy is probably correct, the idea that children who have more infections are less likely to develop allergies is now largely discounted.1

The more recent finding which highlights the importance of this issue is the growing evidence that this concept applies not just to allergies, but to a much broader range of chronic inflammatory diseases (CIDs) such as type 1 diabetes, multiple sclerosis and inflammatory bowel disease, and also to some types of depression and cancer.

In 2003 Graham Rook proposed the “Old Friends Mechanism” which seems to offer a more rational explanation (reviewed in 3 recent papers2,3,4). He argues that the vital exposures are not colds, influenza, measles and other common childhood infections (as originally proposed by Strachan) which have evolved relatively recently, over the last 10,000 years, but rather the microbes already present over 2 million years ago in hunter gatherer times when the human immune system was developing. His argument is that we have become so dependent on these Old Friends that our immune systems cannot function properly without them. Although these microbes are still abundant, it appears that we (particularly those living in the industrialised countries of the developed world) have lost touch with these Old Friends, through a variety of lifestyle, public health and medical changes.

Rook suggests that our “Old Friends” are most likely to include: Environmental species which inhabit our indoor and outdoor environments The normal microbiota of the human skin, gut and respiratory tract, and that of the

animals we live with Organisms such as hepatitis A virus and helminths (worms) which establish chronic

infections or carrier states, and that need to be tolerated. These are usually referred to as the “Old infections”. 2,3,4,5

Studies are now showing how exposure to these microbes is vital because they interact with the regulatory system that keeps our immune system in balance. Without this the immune system may react inappropriately, sometimes overreacting to cause asthma and hay fever, sometimes attacking our own tissues to cause autoimmune diseases such as multiple sclerosis.2,3 Relevant to the discussion about the possible role of antibiotics is the evidence

which suggests that diversity of exposure to these microbes is key to building a well regulated immune system.

For allergic disease, it seems that the most important times for exposure are early in development, during pregnancy and the first few days or months of infancy, and that exposure needs to be maintained over a significant period. This fits with evidence that Caesarean section may be associated with increased tendency to allergies, whilst breastfeeding for six months or more can be protective. There is some evidence that exposures need to be maintained during childhood and adult life, but more research is required to better understand.

What has caused the problem and why now?

Since allergies and other CIDs are largely diseases of the last 100 years or so, the obvious assumption from the original hygiene hypothesis proposition was that the “sanitary” revolution of the last 200 years is a route cause. During this time we saw radical improvements to sanitation, cleaner food and water, the clean-up of city streets and a decline in infectious diseases through reduced exposure to infectious disease agents. The link to the sanitary revolution may be correct, but, if the OF mechanism is correct, the likely explanation is that these changes have also inadvertently reduced exposure to our Old Friends which occupy the same habitats.

von Hertzen6 and Rook4 argue that, ultimately, reduced exposure to important microbial species has happened largely because of changes in, and reduced contact with, our outdoor environment and the huge diversity of microbial and helminth species which it contains. Urbanisation has accelerated this loss of exposure to the natural environment. This is in line with studies which consistently show that exposure to a farm environment early in life can protect from asthma. In turn the composition of the human commensal microbiota depends on input from the natural environment. Hanski et al7 showed that the skin microbiota from individuals living close to agricultural land in Finland was more diverse than that from individuals living close to urban centres, and was associated with reduced atopic sensitisation. We also have different diets and lifestyles which affects the nature of our gut, skin and respiratory microbiota.

In his original 1989 hypothesis Strachan suggested that a cause of declining microbial exposure could be “improved household amenities and higher standards of personal cleanliness”. From this the notion that “we have become too clean for our own good” has arisen. If home and personal cleanliness contributes per se, its role is likely to be small. The key point may be that the microbial content of modern urban homes has altered because mostly our homes now interact with urban environments and are populated by people with different and less diverse human microbiota. We now interact with an altogether different and less diverse mix of microbes relative to earlier generations which were largely rural. These issues are reviewed in more details by Stanwell Smith, Bloomfield and Rook 20128,9

Increasingly there is evidence that antibiotic use may be an important contributory factor. This was first proposed in 1999.10 Since 1999 a growing number of epidemiological studies have shown that antibiotics, particularly excessive antibiotic use during pregnancy or the neonatal period, induces changes in the microbiota, and is associated with increased risk of asthma, cow’s milk allergy and irritable bowel disease (IBD). A potential explanation is that broad spectrum antibiotics alter the gut microbiota, which in turn affects the maturing immune system in a way that promotes allergic disease development. However much further work is required to better understand the mechanisms whereby antibiotics exert adverse effects on the gut microbiota and the development of asthma.

It is notable that antibiotic usage trends, (although also trends for many other drugs), show a fairly good temporal fit with the critical rise in allergies and CIDs documented since the 1970s. Antibiotic use increased rapidly after the development of penicillin in the 1940s and discovery of other antibacterial agents. Widespread use preceded the rapid rise of atopic disorders from the 1970s and has continued to increase, although from the 1990s, increasing concern about antibiotic resistance has led to restrictions on prescribing some types of antibiotics (Working Party Report 199411) and decline in usage in some countries, such as France and Japan (Hamad 201012). In their 2012 review, Stanwell Smith et al10,11

concluded that antibiotic usage trends show a better fit, compared with those for water and sanitation improvements, with the rise in allergies and CIDs documented since the 1970s.

Although an association has been demonstrated between antibiotic use and later development of asthma or allergy in a larger number of epidemiological studies, other studies have failed to find a link. Still other studies suggest that it is due to frequent antibiotic use in early life, which is more common amongst asthmatic children.

Are other factors involved?

Although it now seems clear that human:microbe interactions are a fundamental factor, the risk of allergic and other CIDs depends on a whole range of factors such as changes in diet, pollution, physical activity, obesity, socio-economic factors and stress. Genetic predisposition is also a factor. This could explain why we do not all suffer from these diseases. We all still get some microbial exposure, which for some is sufficient, for others not. For these people allergies and other CIDs may be triggered, when one or more of these other factors cause further immune dysregulation.

Since there are a multitude of factors which determine the risk to any individual person of developing a CID, and because these factors are often interdependent, it must be borne in mind that, for some individuals, antibiotic use are unlikely to be a risk factor. For example, if the microbiota is already too impoverished, clearly antibiotics will make no difference.  Or, if the important organisms are species which are antibiotic resistant (e.g in their investigations of food allergy, Nagler and co-workers13 have shown, using gnotobiotic mice, that the allergy-protective capacity is conferred by a Clostridia-containing microbiota) then antibiotics will be irrelevant. If there is a critical window in the infant's development (as demonstrated in mice,14) timing of antibiotic exposure in infancy could also be a crucial factor.

In the following sections the various epidemiological and biological studies supporting and questioning the link between antibiotic usage and the development of allergies and other Chronic Inflammatory Diseases are reviewed.

Antibiotic use and asthma and allergies

Early epidemiological studies examining the association between early childhood antibiotic use and later development of asthma or allergy carried out between 1999 and 2006 (Von Mutius 199915, Wickens 199916, Droste 200017, McKeever 200218, Cohet 200419 and Thomas 200620) are reviewed by Sevin and Peebles in 2010.21 Early studies on the link between antibiotic use and allergic disorders in childhood (mainly asthma and excema) is also reviewed by Jordan et al 2008.22 Whilst some studies showed a significant association between antibiotic use and the development of asthma, several of the early studies did not (Celedon 200223 200424, Illi 200125).

Early on, it was suggested that the inconsistent results in some of these early studies may have been due to bias from use of antibiotics to treat lower respiratory tract infection, which is

more common in asthmatics (reverse causation). Frequent antibiotic use in early life is more common amongst asthmatic children (Celadon 200426). However, in the 2007 study by Kozyrskyj27, a significant association of antibiotic use with atopy risk was observed when children with asthma diagnosed before 6 months were excluded and those without lower respiratory infection analysed separately. It was argued that confounders for this effect could also include the use of paracetamol to treat fevers, associated with increased asthma and allergy in some studies, as well as exposure to antibiotics via breastfeeding. It was also suggested that inappropriate prescribing of antibiotics for young children may also complicate interpretation of study results and the effects of antibiotics may also be affected by exposure to antibiotic residues or by-products, which may persist in treated water.

More recent studies (2007-2014), summarised below, consistently support the finding that antibiotic use may be associated with increased risk of childhood asthma, although the issue of reverse causality and other confounding factors is still under debate.28,29 The data also indicate that increased risk of asthma is associated with maternal antibiotic use:

A Canadian birth cohort study by Kozyrskyj et al 2007, of 13,116 children, found that use of antibiotics in the first year of life was associated with increased risk of asthma at age 7 years. The highest risk occurred among children receiving more than four courses of antibiotics in the first year (OR 1.46). A larger 2009 follow-up study by Marra et al 200930 (251,817 children), adjusting for possible confounders such as socio-economic status, birth weight, gestational age, and congenital abnormalities, revealed a small increase (adjusted hazard ratio (HR) 1.12) in the development of asthma in children receiving antibiotics in the first year of life. The risk also increased with the number of antibiotic courses prescribed (adjusted HR for 44 courses 1.30).

Kummeling et al 200731 collected data on antibiotic use in the first 6 months, and eczema and wheeze until age 2 by questionnaires in 2764 families participating in a Netherlands Birth Cohort Study. During the first 2 years, eczema was present in 32% of infants, wheeze in 11%, and prolonged wheezing in 5%. At 2 years, 27% of children were sensitised against at least one allergen. At 6 months, 11% had been exposed to antibiotics through breast milk and 20% directly through medication. Risk of recurrent wheeze (adjOR=2.65, 95% confidence interval: 1.95-3.60) and prolonged wheeze (adjOR=2.32, 95% CI: 1.55-3.48) was higher in infants directly exposed to antibiotics, also after excluding children who wheezed during the period of antibiotic use (avoiding reverse causation). Antibiotic use through breastfeeding was associated with recurrent wheeze (adjOR=1.55, 95% CI: 1.02-2.37), but prolonged wheeze was not (adjOR=1.12, 95% CI: 0.62-2.02). Eczema was not associated with antibiotic use suggesting that different mechanisms may underlie the etiology of wheeze compared to eczema.

Using data from Phase III of the International Study of Asthma and Allergies in Childhood (ISAAC) Foliaki et al 200932 demonstrated an association between the use of antibiotics in the first year of life and symptoms of asthma and also rhinoconjunctivitis, and eczema in children 6 and 7 years old. A total of 193,412 children in 29 countries participated. Reported use of antibiotics in the first year of life was associated with an increased risk of current asthma symptoms (wheezing in the previous 12 months) with an OR (adjusted for sex, region of the world, language, and per capita gross national income) of 1.96 (95% CI, 1.85-2.07); this fell to 1.70 (1.60-1.80) when adjusted for other risk factors for asthma. Similar associations were observed for severe asthma symptoms (OR, 1.82; 95% CI, 1.67-1.98), and asthma ever (OR, 1.94; 95% CI, 1.83-2.06). Use of antibiotics in the first year of life was also associated, but less strongly, with increased risks of current symptoms of rhinoconjunctivitis (OR, 1.56; 95% CI, 1.46-1.66) and eczema (OR, 1.58; 95% CI, 1.33-1.51).

In 2011, Penders, Kummeling and Thjis28 carried out a systematic review and meta-analysis longitudinal study on antibiotic use and subsequent development of wheeze and/or asthma with regards to study quality, outcome measurement, reverse causation (RC; wheezing/asthma symptoms have caused prescription of antibiotics) and

confounding by indication (CbI; respiratory tract infections leading to antibiotic use may be the underlying cause triggering asthma symptom development). The authors concluded that there has been overestimation of an association between antibiotic use and subsequent wheezing or diagnosis of asthma, possibly due to varying definitions of disease. The study evaluated 21 longitudinal studies. The effect of antibiotic use on wheeze/asthma risk varied between studies. 18 studies were eligible for meta-analysis showing pooled OR 1.27 (95% CI 1.12-1.43) for wheeze/asthma. When studies with possible RC and CbI were eliminated, the pooled risk estimate in the nine remaining studies was attenuated to OR 1.12 (95% CI 0.98-1.26). Definition of wheeze/asthma and age at follow-up differed between studies. Three studies focused on wheeze/asthma beyond 5-6 yrs of age with the presence of active symptoms and/or medication (pooled OR 1.08, 95% CI 0.93-1.23; dominated by one study). RC and CbI lead to overestimation of the association between antibiotic use and subsequent development of wheeze/asthma. Association was weak when fully adjusted for these types of bias. Heterogeneity of disease definition between studies could affect the results

In 2011 Risnes et al33 reported a cohort study (2003-2007) of 1,401 US children which showed that antibiotic use within the first 6 months of life was associated with increased risk of asthma at 6 years (adjusted odds ratio1.52, 95% confidence interval (CI): 1.07, 2.16). The odds ratio if asthma was first diagnosed after 3 years of age was 1.66 (95% CI: 0.99, 2.79) and, in children with no history of lower respiratory infection in the first year of life, the odds ratio was 1.66 (95% CI: 1.12, 3.46). The adverse effect of antibiotics was particularly strong in children with no family history of asthma (odds ratio 1.89, 95% CI: 1.00, 3.58). The odds ratio for a positive allergy blood or skin test was 1.59 (95% CI: 1.10, 2.28). The authors concluded not only that early antibiotic use was associated with asthma and allergy at 6 years of age, but also that protopathic bias (antibiotics used to treat early symptoms of asthma) was unlikely to account for the main findings.

Almqvist et al 201134 carried out a study of the association between different classes of antibiotics and prescription of asthma medication in a cohort of all Swedish children, born between July 2005 and June 2009, ever treated with antibiotics. In total, 211,192 children had received prescriptions of antibiotics. There was a strong association between prescription of antibiotics and prescription of asthma medication. The hazard ratios (HRs) for asthma medication associated with prescription of amoxicillin, penicillin, cephalosporin and macrolides (Gram-positive infections) were stronger than HRs associated with prescription of sulphonamides, trimethoprim and quinolones (urinary tract infections) and flucloxacillin (skin and soft tissue infections), e.g. first year HR = 2.27 (95% confidence intervals 2.17–2.37) as compared with HR = 1.04 (0.78–1.40). The HR for broad spectrum antibiotic use was significantly higher than for narrow spectrum. The authors concluded however that the association is subject to reverse causation or confounding by indication due to respiratory tract infections.

Murk et al 201129 carried out a study of the association between antibiotic exposure during pregnancy or in the first year of life and risk of childhood asthma using studies published between 1950 and 2010. Those that assessed associations between antibiotic exposure during pregnancy or in the first year of life and asthma at ages 0 to 18 years (for pregnancy exposures) or ages 3 to 18 years (for first-year-of-life exposures) were included. Validity was assessed according to study design, age at asthma diagnosis, adjustment for respiratory infections, and consultation rates. For exposure in the first year of life, the pooled odds ratio (OR) for all studies (N=20) was 1.52 (95% confidence interval [CI]: 1.30-1.77). Retrospective studies had the highest pooled risk estimate for asthma (OR: 2.04 [95% CI: 1.83-2.27]; n=8) compared with database and prospective studies (OR: 1.25 [95% CI: 1.08 -1.45]; n=12). Risk estimates for studies adjusted for respiratory infections (pooled OR: 1.16 [95% CI: 1.08-1.25]; n= 5) or later asthma onset (pooled OR for asthma at or after 2 years: OR: 1.16 [95% CI: 1.06-1.25];n=3) were weaker but remained significant. For exposure during pregnancy (n=3), the pooled OR was 1.24 (95% CI: 1.02–1.50). The authors concluded that antibiotics slightly increase the

risk of childhood asthma but reverse causality and protopathic bias are possible confounders.

Jedrychowski et al 201135 studied the association between the use of various classes of antibiotics (penicillin, cephalosporin and macrolide derivatives) in early childhood and the medical diagnosis of asthma or wheezing reported by mothers over the follow-up after adjustment for potential confounders and respiratory infections. In a population-based sample of 5-year-olds, a part of the ongoing birth cohort study, the standardized interviews on health outcomes, potential confounders (child’s gender, maternal atopy, parity, prenatal and postnatal environmental tobacco smoke) and the use of antibiotics were gathered from mothers of 310 children. While the overall use of antibiotics during the early childhood was insignificantly associated with asthma (adjusted OR = 1.65, 95%CI: 0.93–2.93), risk estimates were significant both for macrolide antibiotics (adjusted OR=2.14, 95%CI: 1.16–3.95) and cephalosporins (OR=1.98, 95%CI: 1.14–3.37). The significant excess in IRR (incident risk ratio) of wheezing episodes was related only to the use of macrolide antibiotics (adjusted IRR=1.91, 95%CI: 1.12–3.27). The use of other classes of antibiotics was found not to be associated with the medical diagnosis of asthma or wheezing episodes recorded in the study period.

Raciborski et al 201336 carried out a study based on the ISAAC questionnaire which suggested a correlation between the use of antibiotics in the first 3 years of life and asthma and allergy symptoms in children aged 6-8 years. Number of courses of antibiotics was also found to be an important factor. The study was survey-based with a self-completed questionnaire. The respondents were parents of children aged 6-8 years living in Warszawa, Poland. 1461 completed questionnaires were collected. Asthma was declared in 4.3% of children. Wheezing and/or sibilant rhonchi within 12 months before the study was observed in 13.5% of cases. Asthma medication was taken by 21.8% of children. Allergic rhinitis was declared in 18.7% of the children. Problems with sneezing, rhinorrhea, and nasal congestion not associated with cold or fever were observed in 40.7% of the children. The analysis of the odds ratios between the use of antibiotics and the symptoms of allergic diseases revealed a clear correlation. The highest odds ratio was observed between the completion of over three courses of antibiotic therapy prior to the age of 12 months and the declaration of one of the following: asthma (OR = 5.59, 95% CI: 2.6-12.01), wheezing and/or sibilant rhonchi (OR = 4.68, 95% CI: 3.01-7.27) and taking medicines for breathlessness (OR = 5.12, 95% CI: 3.42-7.68).

Stensballe et al 201337 investigated whether increased risk of asthma was associated with maternal antibiotic use in a birth cohort with increased risk of asthma. The findings were replicated in an unselected national birth cohort, and in a subgroup using antibiotics for non respiratory infections. Subjects were included from the Copenhagen Prospective Study on Asthma in Childhood cohort of children born of mothers with asthma (N = 411). The study showed increased risk of asthma exacerbation (hazard ratio 1.98 [95% CI 1.08-3.63]) if mothers had used antibiotics during third trimester. The Danish National Birth Cohort confirmed increased risk of asthma hospitalization (hazard ratio 1.17 [1.00-1.36]), and inhaled corticosteroids (1.18 [1.10-1.27]) in the children if mothers used antibiotics during pregnancy. In the subgroup of mothers using antibiotics for non respiratory infection, the children also had increased risk of asthma.

Taskok et al 201338 carried out a systematic review and meta-analysis of observational studies, involving children or young adults aged 0–25 years, demonstrating the impact of antibiotic exposure during the first 12 months of life, but not in utero, on subsequent eczema risk. Twenty studies examined the association between prenatal and/or postnatal antibiotic use and development of eczema. The pooled OR for the 17 studies examining postnatal antibiotic exposure was 1·41 [95% confidence interval (CI) 1·30–1·53]. The pooled OR for the 10 longitudinal studies was 1·40 (95% CI 1·19–1·64), compared with a pooled OR of 1·43 (95% CI 1·36–1·51) for the seven cross-sectional studies. There was a significant dose–response association, suggesting a 7% increase in the risk of eczema for each additional antibiotic course received during the first year of life [pooled OR 1·07

(95% CI 1·02–1·11)]. Finally, the pooled OR for the 4 studies of antenatal exposure was 1·30 (95% CI 0·86–1·95). They concluded that exposure to antibiotics in the first year of life, but not prenatally, is more common in children with eczema.

Hoskin-Parr et al 201339 investigated association of antibiotic use in the first 2 yr of life with development of asthma, eczema or hay fever by age 7.5 yr in a birth cohort of 4952 children from the Avon Longitudinal Study of Parents and Children (ALSPAC). A robust and dose-dependent association was found between antibiotic use in the first 2 yr of life and asthma at age 7.5 yr Children reported to have taken antibiotics during infancy (0–2 yr) were more likely to have asthma at 7.5 yr (OR 1.75, 95% CI 1.40–2.17), and the odds (OR, [95% CI]) increased with greater numbers of courses: once 1.11 [0.84–1.48]; twice 1.50 [1.14–1.98]; three times 1.79 [1.34–2.40]; four times or more 2.82 [2.19–3.63]. Increased antibiotic use was also associated with higher odds of eczema and hay fever. Skin prick tests at age 7.5 yr suggested that, although increased antibiotic use was associated with higher odds of asthma, eczema and hay fever, it did not appear to be mediated through an association with atopy. The effect appeared to be associated with cumulative rather than a critical period of exposure during the first 2 yr.

Using medical records Semic-Jusufagic et al 201440 investigated the association between antibiotic prescription and subsequent development of atopy, wheeze, and asthma exacerbation in a birth cohort of 916 children followed from birth to age 11 years. Higher risk of physician-confirmed wheezing was recorded after antibiotic prescription (hazard ratio [HR] 1·71, 95% CI 1·32—2·23; p<0·0001) and severe wheeze or asthma exacerbation after antibiotic prescription (HR 2·26, 95% CI 1·03—4·94; p=0·041). In children who wheezed, the hazards of exacerbations (2·09, 1·51—2·90; p<0·0001) and admissions to hospital (2·64, 1·49—4·70; p=0·0009) were significantly increased in the 2 years after the first antibiotic prescription. Assessment of responses of peripheral blood mononuclear cells, taken at age 11 years showed that children who received antibiotics in infancy had significantly lower induction of cytokines, which are important in host defence against virus infections to both RSV and rhinovirus; there were no differences in antibacterial responses (Haemophilus influenzae and Streptococcus pneumoniae). Variants in 17q21 were associated with an increased risk of early life antibiotic prescription. The authors concluded however that the associations might arise through a complex confounding by indication. They suggested that hidden factors that may increase the likelihood of both early life antibiotic prescription and later asthma are an increased susceptibility to viral infections consequent upon impaired antiviral immunity and genetic variants on 17q21.

Metsala et al 201441 report a study showing that both prenatal and postnatal exposure to antibiotics was associated with increased risk of asthma. Children (22,000), born in 1996–2004 in Finland and diagnosed with asthma by 2006, were identified from a national health register. For each case, a matched control was selected. Information on asthma diagnoses, anti-asthmatic drugs and antibiotics, as well as putative confounders was obtained from national health registries. Maternal use of any antibiotics during pregnancy was associated with increased risk of asthma in the offspring (adjusted odds ratio [OR]=1.31 [95% confidence interval [CI]: 1.21-1.42]). Several maternal specific antibiotics were associated with risk of asthma and the strongest association was for cephalosporins (OR1.46 [95% CI 1.30-1.64]). Child's use of antibiotics during the first year of life was associated with increased risk of asthma (OR = 1.60 [95% CI 1.48–1.73]). Child's use of cephalosporins (OR1.79 [95% CI 1.59-2.01]), sulphonamides and trimethoprim (OR1.65 [95% CI 1.34–2.02]), macrolides (OR = 1.61 [95% CI 1.46–1.78]) and amoxicillin (OR1.46 [95% CI 1.35-1.58]) was associated with increased risk of asthma.

Compared with postnatal exposure, prenatal exposure to antibiotics is less well studied,42,43,44,45,46,47,48 but most studies suggest an increased risk in the offspring.43,44,46,47,48

As the use of antibiotics by the mothers during pregnancy was not extensive, the observed association between maternal antibiotics and the risk of asthma in the offspring may not

have such great public health importance. On the contrary, as the use of antibiotics during early childhood is common, the public health importance of the association between child’s use, if confirmed in future studies, may be more significant.

Antibiotic use and other allergic and chronic inflammatory diseases

Recent studies are now showing that antibiotics, particularly excessive antibiotic use during pregnancy or the neonatal period are also implicated in other allergic and inflammatory diseases such as cow’s milk allergy, irritable bowel disease (IBD – including Crohn’s disease and ulcerative colitis) and irritable bowel syndrome (IBS):

Metsala et al 201349 investigated the associations between mother's and offspring's use of antibiotics and the risk of cow's milk allergy in infancy. Using a national registry, children who were born in 1996-2004 in Finland and diagnosed with cow's milk allergy after 1 month of age by November 2005 (n = 15,672) were identified. For each case, one control matched for birth date, sex, and hospital district was selected. Maternal use of antibiotics before and during pregnancy was associated with an increased risk of cow's milk allergy in the offspring (odds ratio = 1.26 [95% CI = 1.20-1.33] and 1.21 [1.14-1.28], respectively, adjusting for putative confounders). The risk of cow's milk allergy increased with increasing antibiotics used from birth to diagnosis (test for trend P <0.001).

Five studies carried out in 2011/2 reported potential association between use of antibiotics and IBD or IBS in childhood:

Shaw et al 201150 carried out a study which showed that use of antibiotics 2–5 years before diagnosis was associated with the development of IBD (crohn’s disease and ulcerative colitis). This was a nested case–control analysis of the population-based University of Manitoba Inflammatory Bowel Disease Epidemiologic Database. A total of 2,234 subjects diagnosed with IBD between 2001 and 2008 were matched to 22,346 controls. The mean age at diagnosis was 43.4 years. In all, 12% of cases had ≥3 prescriptions 2 years before the case date, compared with 7% of controls. The odds ratio for those receiving ≥3 dispensations 2 years before their study inclusion was 1.5 (95% confidence interval: 1.3,1.8; P<0.0001) of being an IBD case. This difference in ≥3 dispensations between cases and controls was fairly consistent at 3, 4, and 5 years before IBD case date. Antibiotic dispensations were associated with both Crohn's disease (CD) and ulcerative colitis (UC), with the association nominally stronger in CD cases for ≥1 and ≥2 dispensations, while the association was stronger in UC cases for ≥3 dispensations. A dose-dependent relationship between number of antibiotic courses, and risk of IBD was observed across all years investigated. This follows an earlier 2010 investigation which also showed an association between antibiotic use and IBD.51

Hviid et al 201152 carried out a study to evaluate the potential association between use of antibiotics and IBD in childhood. A nationwide cohort study was conducted of all Danish singleton children born from 1995 to 2003 (N=577 627) with individual-level information on filled antibiotic prescriptions, IBD and potential confounding variables. IBD was diagnosed in 117 children during 3 173 117 person-years of follow-up. The RR of IBD was 1.84 (95% CI 1.08 to 3.15) for antibiotic users compared with non-users. This association appeared to be an effect on Crohn's disease (CD) alone (RR 3.41) and was strongest in the first 3 months following use (RR 4.43) and among children with ≥7 courses of antibiotics (RR 7.32).

Villarreal et al 201253 investigated the associations between IBS and exposure to broad-spectrum antibiotics. Medical records of adults who were started on a broad-spectrum antibiotic at Gundersen Lutheran Health System (USA) between January 1, 2008, and December 31, 2008, were reviewed retrospectively. From this population, those who developed IBS within 12 months were identified and compared their demographic and

clinical characteristics with the characteristics of those who did not. Of the 26,107 adult patients exposed to broad-spectrum antibiotics during the study period, 115 received an IBS diagnosis within 12 months. Most were women (84 %; n = 97), and they had a higher prevalence of associated co-morbidities than those who did not develop IBS. Patients indicated for macrolide or tetracycline use had a higher proportion of IBS development within 12 months; indication for tetracycline use maintained significance even after controlling for sex and comorbid conditions (odds ratio; 1.48; P = .046).

Kronman et al 201254 carried out a study demonstrating an association between childhood anti-anaerobic antibiotic exposure and IBD development. This retrospective cohort study employed data from 464 UK ambulatory practices. All children with ≥2 years of follow-up from 1994 to 2009 were followed between practice enrollment and IBD development, practice deregistration, 19 years of age, or death. Anti-anaerobic antibiotic agents were defined as penicillin, amoxicillin, ampicillin, penicillin/β-lactamase inhibitor combinations, tetracyclines, clindamycin, metronidazole, cefoxitin, carbapenems, and oral vancomycin. A total of 1 072 426 subjects contributed 6.6 million person-years of follow-up; 748 developed IBD. IBD incidence rates among antianaerobic antibiotic unexposed and exposed subjects were 0.83 and 1.52/10 000 person-years, respectively, for an 84% relative risk increase. Exposure throughout childhood was associated with developing IBD, but this relationship decreased with increasing age at exposure. Exposure before 1 year of age had an adjusted hazard ratio of 5.51 (95% confidence interval [CI]: 1.66–18.28) but decreased to 2.62 (95% CI: 1.61–4.25) and 1.57 (95% CI: 1.35–1.84) by 5 and 15 years, respectively. Each antibiotic course increased the IBD hazard by 6% (4%–8%). A dose-response effect existed, with >2 antibiotic courses more highly associated with IBD development than 1 to 2 courses, with adjusted hazard ratios of 4.77 (95% CI: 2.13–10.68) versus 3.33 (95% CI: 1.69–6.58).

Virta et al 201255 conducted a national, register-based study of children born in 1994–2008 in Finland and diagnosed with IBD by October 2010. The authors identified 595 children with IBD (233 with Crohn’s disease and 362 with ulcerative colitis) and 2,380 matched controls matched. Risk of pediatric Crohn’s disease increased with the number of antibiotic purchases from birth to the index date and persisted when the 6 months preceding the case’s diagnosis were excluded (for 7–10 purchases vs. none, OR=3.48, 95% confidence interval: 1.57, 7.34; conditional logistic regression). The association between Crohn’s disease and antibiotic use was stronger in boys than girls (P=0.01). Cephalosporins showed the strongest association with Crohn’s disease (3 purchases vs. non use, OR=2.82, 95% confidence interval: 1.65, 4.81). Antibiotic exposure was not associated with development of pediatric ulcerative colitis.

Current assessments of evidence for association between pre and postnatal antibiotic exposure and development of asthma

In reporting the findings of their 2013 and 2014 study, Metsala et al41 and Hoskin-Parr et al39 present a useful discussion of their findings and conclusions, in relation to other currently existing data, on the association between pre and postnatal exposure to antibiotics and the development of asthma and allergies. They also discuss the strength of their data in relation to possible confounding factors, and in relation to both their own and earlier studies.

1. Key observation and conclusions made by Metsala et al41 on their study of 22,000 children in Finland are as follows:

In general, evidence indicating the role of antibiotics during the pre and postnatal period in development of childhood asthma is accumulating, although fewer studies have focused on prenatal antibiotics, where increased risk was observed in some studies,

while null associations were reported in 2 studies. Although there is now well established and extensive epidemiological evidence on postnatal antibiotic exposure and risk of asthma or wheeze, it is still regarded as inconclusive by some experts. The largest cohort studies consistently report an increased risk of asthma in children exposed to antibiotics in early childhood (Mckeever 200246, Almqvist 201234, Marra 200630, Kozyrskyj 200727, Martel 200943 Metsala 201441).

Evidence on the role of specific antibiotics in the development of asthma is limited and inconsistent. No specific antibiotic used by mothers has been reported to be associated with asthma in the offspring. The child’s use of macrolides, cephalosporins, amoxicillin, penicillin and broad-spectrum antibiotics has been associated with increased risk of asthma. In accordance with most previous studies, Metsala et al recorded variation in the strength of the associations between different antibiotics. In contrast to the study by Almqvist34, Metsala et al found that estimates for antibiotics against urinary tract infections, although statistically non-significant, were close to effect estimates for antibiotics against respiratory tract infections. This inconsistency may be due to differences in antibiotic prescription practices between countries and differences in methodology, such as asthma definition and follow-up time.

With regard to the issues of reverse causality (asthma symptoms are responsible for the prescription of antibiotics) or confounding by indication (respiratory infections may serve an indication for antibiotic prescribing and as a risk factor for asthma), Metsala et al report that direct associations have been observed also in studies taking infections into account (Risnes33, Marra30, Martel43, Korzyskyj 200727, McKeever46). Further, the associations were even stronger when those children who had respiratory infections were excluded (Risnes33, Marra 30).

Metsala et al also make further observations about their own large, population and register-based study in Finland:

With regard to confounding by indication, Metsala concluded that, although the statistically non-significant associations between antibiotics for urinary tract infection and asthma suggest that confounding by indication could partly explain their findings, the role of low number of observations in the analysis resulting in wide 95% confidence intervals should be taken into account as an explanation for the non-significant findings. Further, they observed an increased risk of asthma also in children who were diagnosed with asthma first time at the age of 6 years or later, indicating that reverse causality is not a major form of bias in their data.

When extending the child's exposure period beyond the first year of life, stronger associations than previously reported were observed.

Both prenatal and postnatal antibiotic exposure was associated with increased risk of asthma in childhood in a dose-related manner. Several commonly used antibiotics were associated with the risk of asthma. Increased risk of asthma was observed not only with antibiotics commonly used for respiratory infections, but also antibiotics for urinary tract infections, indicating no clear evidence of confounding by indication.

They stated however “One of the limitations of our study is that despite including several putative confounders

we missed information on indication of antibiotic prescription, exposure at hospitals and eczema as well as certain factors affecting the composition of the gut’s microbiota, such as infant feeding and the use of pre- and probiotics. Thus, residual confounding as an explanation of the observed results cannot be ruled out. Further, despite having a large data some of our sub-group analyses suffered lack of power.

2. The main strength of the birth cohort study by Hoskin-Parr et al 201339 of 4952 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) was the use of a large longitudinal cohort, with prospective collection of exposure data and the ability to account for

a wide range of confounding influences. The availability of symptom reports in infancy allowed investigators to test for reverse causation of antibiotic prescription for allergic symptoms. Investigators were also able to test for dose dependency and evaluate whether timing of antibiotics was linked to outcome. As data was collected within 12 months of the event, there was also less likelihood of recall bias. Key observation and conclusions made by Hoskins-Parr et al are as follows:

Results showed a strong, dose-dependent association between antibiotics in infancy and asthma in later childhood. Although the association was weaker with eczema and hay fever, associations were not attenuated by adjustment for confounders.

Analysis of timing and number of courses of antibiotics suggested that cumulative exposure may have been more important than a single critical period effect

The authors concluded that, although they found an association between reported asthma and antibiotic use in infancy that appeared to be robust to confounding, they could not completely discount the possibility of confounding by indication and reverse causation.

The fact that exclusion of children with early wheeze up to 30 months, for which they may have received inappropriate antibiotics, weakened the association substantially, it must be considered as evidence that reverse causation explained part of the association with asthma. This is in line with other studies, as cited above, in which it was possible to document indications for antibiotic prescription, and which suggest that reverse causation or confounding by indication could explain a large part of the reported association. Rusconi et al.47 in 2011 reported an association between antibiotics and early asthma but not with late onset asthma, implying reverse causation. In a longitudinal study of over 4000 subjects, Celedon et al.24 in 2004 reported no association between antibiotic use in the first year of life and asthma development between the ages of 2 and 5 yr when adjusted for lower respiratory illnesses in the first year, indicating confounding by indication.

However - whereas some of the earlier studies were challenged on the basis that they were retrospective studies, with data on antibiotic use dependent on maternal recall up to 6 yr later, which is likely to introduce recall bias33,32, the positive association of asthma and with antibiotic use in this and other prospective studies30 suggest that recall bias alone is insufficient to explain this relationship.

The authors highlighted 2 limitations of their study: The main limitation was reliance on maternal report of antibiotic use, which was not

verified by medical records and which did not specify which antibiotics were used or their indication. Therefore, they were unable to consider confounding by indication. By relying on maternal report for both exposure and outcome, results could be biased by general increased reporting of all health outcomes, including antibiotic use, in questionnaire responses. There is also evidence that a variety of drugs reported by mothers to be used during pregnancy, including antibiotics are associated with asthma risk in their children but the association is largely confounded by concomitant use of anti-asthma drugs by the mother. The authors attempted to account for reporting bias by mothers with asthma, who may be more likely to seek treatment for their children, by considering both reverse causation and by using an unrelated health outcome (headache) as a marker of general health reporting.

A further limitation was that the investigators were unable to categorize antibiotics by class. Jedrychowski et al.35 reported that early use of macrolides and cephalosporins, two broad spectrum antibiotics, was strongly associated with increased risk of asthma at 5 yr of age. In contrast, there is evidence that macrolide antibiotics could increase symptom-free days in children following exacerbations of asthma, possibly through anti-inflammatory effects rather than perturbation of the human microbiome.56 It is likely in UK

practice that broad spectrum antibiotics would have been utilized in this population at the time (early 1990s).

3. The association between antibiotic use and atopy

A further concern expressed by Hoskin-Parr et al39 is that, in contrast to allergy, infant antibiotic use was not positively associated with skin test sensitization at age 7.5 years, indicating no objective evidence of association with atopy.

In their 2008 review paper, Jordan et al22 review a number of studies which linked antibiotics to asthma or eczema, but found no association between antibiotics and atopy as assessed by skin prick tests or measurement of IgE antibodies in venous blood. This is consistent with a more recent reported associations of reduced gut microbial diversity in early infancy and the development of eczema but not atopy in a population of children at high risk of allergy.57

In their 2007 epidemiological study on early antibiotic use, Kummeling et al31 reported an association with eczema until age two but not wheeze. Venous blood samples taken from 815 infants at age 2 years was also analyzed for total and specific IgE against common food and inhalant allergens, but the data suggested that sensitisation was not associated with antibiotic exposure The authors suggested that different biological mechanisms may underlie the etiology of wheeze compared to eczema or sensitisation.

These observations are contradictory to the putative mechanism of association of allergies with antibiotic use, thought to involve pressure on the developing immune system towards an atopic phenotype. It suggests that other mechanisms may also be involved including other mechanisms which may mediate effects of alterations in gut microbiota, such as non-atopic inflammation.

Biological plausibility: evidence from human and animal studies

The potential explanation for the association between antibiotic usage and CIDs is that antibiotics alter the microbiota of the human body, particularly during the critical period for immune development, which in turn affects the immune system in a way that predisposes to development of inflammatory disease. Russell and Murch 200658 propose that the mechanism by which prenatal and postnatal antibiotics could influence development of asthma is their adverse and possible long-term effect59 on gut microbiota of both the mother and the child and vaginal microbiota of the mother In support of this, Faa 201360 and Westerbeek 200661 found that antibiotic use in the perinatal period delays colonization by Bifidobacter and Lactobacillus species.

Whereas human epidemiological studies have been revealing, due to ongoing concerns about confounding variables, animal studies are being increasingly used to better understand the impact of antibiotics on the development of allergic disease. These studies are beginning to reveal the mechanisms by which this may occur, and importantly to reveal that it is the altered microbiota which is associated with increased risk of inflammatory diseases, not the antibiotic per se.

There is now a considerable amount of biological data involving humans and experimental animals which indicates that changes per se in the microbiota are implicated in allergic and other inflammatory diseases. This data is reviewed by Reynolds and Finlay62 and Rook et al.2,3.4 In the following sections we review some of the animal and human studies which

have shown how excessive antibiotic usage at key life stages is associated with alteration in human microbiota and development of allergic and other diseases. This aspect is also discussed in the reviews by Reynolds and Finlay62 and Rook et al.2,3,4

Russell et al 201263 used neonatal mice to study early life antibiotic-driven changes in microbiota and susceptibility to allergic asthma. Mice exposed to clinically relevant doses of vancomycin in utero via their mother’s drinking water, and throughout life, had profound shifts in their fecal microbiota communities, which become dominated by Lactobacillaceae. The mice exhibited high serum IgE levels, and enhanced disease severity in an ovalbumin-driven model of allergic asthma, evidenced by worse lung pathological scores and increased airway hyperresponsiveness. Neither antibiotic had a significant effect when administered to adult mice. Noverr et al 200564 demonstrated a similar effect in mice, with altered immune regulation in the airways and up-regulation of Th2 responses to allergen observed after a short course of broad-spectrum antibiotics.

The persistence of antibiotic-induced perturbations of the microbiota appears to depend on the class of antibiotic being administered. Robinson et al 201065 found that some antibiotics used to treat mice produced significant changes in intestinal microbiota composition but bacterial communities returned to baseline after withdrawal of antibiotics. By contrast, for others such as cefoperazone-treated mice the diversity of microbes was still quite disrupted even 6 weeks following the end of antibiotic treatment. Reynolds and Finlay62 suggest that, if it is confirmed that certain classes of antibiotic are associated with a heightened risk of disease development, then the prescription of these drugs can be avoided where other lower-risk antibiotics are available.

Antibiotic treatment in adult mice has also been linked to exacerbations in allergic disease in adulthood. Russell and Murch 200658 found that, when adult mice (at least 8 weeks old) were treated with a broad-spectrum cocktail of antibiotics, including ampicillin, gentamicin, metronidazole, neomycin and vancomycin for 4 weeks, serum levels of IgE are elevated, along with circulating basophil numbers. From their review of this issue Reynolds and Finlay62 conclude “An important area for future focus will be to characterize those immune disruptions following early-life antibiotic treatment that persist into adulthood, and to confirm what the critical window for recovery of these perturbations is. Probiotic treatment in humans has shown little promise to date in reducing the risk of allergy development, however it is possible that a beneficial effect would be seen, if the correct mix of bacteria are administered at the time of antibiotic-mediated microbiota disruption”.

Because of the difficulty in analyzing respiratory microbiota populations, few studies have assessed how antibiotics given orally affect experimental mice, and to what extent these communities can affect allergic airway disease. It has been proposed that immune responses between mucosal surfaces, such as the intestines and lung, are linked, by an as yet unclear mechanism.64 A major challenge for examining the effects of antibiotic treatment is to distinguish between the systemic effects of the intestinal microbiota, versus local impacts of the respiratory microbiota.

Cox et al 201447 have been carrying out studies with mice which suggest that altering the intestinal microbiota by administration of low dose penicillin during a critical developmental window has lasting metabolic consequences. They have shown that low-dose penicillin (LDP), delivered from birth, induces metabolic alterations and affects ileal expression of genes involved in immunity. LDP that is limited to early life transiently perturbs the microbiota, which is sufficient to induce sustained effects on body composition, indicating that microbiota interactions in infancy may be critical determinants of long-term host metabolic effects. These studies characterize important variables in early-life microbe-host

metabolic interaction and identify several taxa consistently linked with metabolic alterations including Lactobacillus and Candidatus Arthromitus (segmented filamentous bacteria).

Hill et al 201266 reported both human and animal studies which showed that alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum IgE concentrations, increased steady-state circulating basophil populations and exaggerated basophil-mediated TH2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Collectively, the results identify a previously unrecognized pathway through which commensal-derived signals influence basophil hematopoiesis and susceptibility to TH2 cytokine–dependent inflammation and allergic disease.

Acknowledments I would like to thank Dr Johanna Metsala and Professor Graham Rook for reading this manuscript and for their useful comments.

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