**1. Introduction**

The augmented number of dementia patients has been dramatic due to the aging of society in advanced countries. Alzheimer's disease (AD) is the most common type of dementia and accounts for more than half (50–70% depending on the reports) of all dementia. AD is charac‐ terized by a gradual onset by developing neuronal damage and continuing cognitive decline related to stress-induced cell damage in the patient's brain [1], which ultimately causes significant

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

impairment to social and occupational functions. The mean duration from the onset of clinical symptoms to the death of the patient has been reported to be approximately 8.5 years [2].

AD is defined by the presence of plaques and tangles in the brain, thus the gold standard for the diagnosis of Alzheimer's disease (AD) is set by means of the histological examination of brain tissue at autopsy, which is usually done after a patient has died, or, rarely, following brain biopsy. On the other hand, clinical diagnosis of AD during life has been performed with a sensitivity ranging from 70.9% to 87.3% and specificity from 44.3% to 70.8% [3]. It was also reported that dementia is often overlooked in community care settings [4].

For objective diagnostic analysis, several biomarkers are available. The reliable biomarker candidates for AD include brain imaging studies using magnetic resonance imaging (MRI) or positron emission tomography (PET), and proteins in cerebrospinal fluid (CSF). MRI is utilized for structural imaging, PET for molecular imaging of amyloid deposition and fluoro-deoxy-D-glucose (FDG)-PET for metabolic imaging, while measurements of amyloid peptide (Aβ) and TAU protein in cerebrospinal fluid (CSF) are used for quantitative analysis. However, structural changes measured by MRI only become apparent in the late stage of AD. Moreover, structural MRI and FDG-PET images are not direct measures of the core pathological hallmarks of AD. PET imaging is relatively expensive and limited in availability. CSF Aβ and TAU might be nonspecific for AD depending on each case [5]. At present, it can be stated that the most well-characterized and validated biomarkers are Aβ and TAU in CSF: the decrease in Aβ with 42 amino acid residues (Aβ42) and increase in TAU and phosphorylated TAU (P-TAU) has been observed in AD patients in several of studies [6].

An alternative method to the invasive CSF collection and expensive specialized facilities for diagnostic imaging is most desirable. Thus, plasma biomarkers have raised expectations because blood sampling is a much less invasive procedure. Blood-based biomarkers have the potential to overcome access and cost barriers and greatly facilitate advanced neuroimaging and cerebrospinal fluid biomarker approaches. Due to the fact that preanalytical processing shows the largest variation in laboratory testing, there are currently no available standardized preanalytical guidelines. In this review, the primary focus is on the fluid biomarkers, especially blood plasma protein biomarkers, as indicators of AD development together with our study on results of a specific blood plasma candidate.


**5. Animal diseases**

*Chlamydia*, as a host, selectively infects humans, other mammals and birds by colonizing certain systems and causing various health issues. Some of these species are zoonotic and can cause human and animal diseases. In this regard, *C. abortus* and *C. psittaci* are essential. *C. abortus* causes abortion, while *C. psittaci* causes a respiratory disease known as psittacosis [13].

The *Chlamydia* diseases in animals that have been confirmed to be transmitted to humans to date are Ovine Enzootic Abortion, and Avian Chlamyiosis. All disease factors are listed below.

#### **5.1 Clamidya abortus at Ovine**

*C. abortus* is responsible for causing Ovine enzootic abortion, a disease that is encountered in almost every region of the world. It is a major factor in lamb losses in sheep herds. Countries that engage in sheep breeding, particularly in Europe, have recognized *C. abortus* as one of the responsible factors for these losses [7, 8].

After 1 or 2 weeks of sheep contracting the disease, discharge related to birth disappears. Pregnant animals that have had a miscarriage due to this disease will not be affected by the disease again. They will not encounter another abortion due

#### *Chlamydias as a Zooonosis and Antibiotic Resistance in Chlamydiae DOI: http://dx.doi.org/10.5772/intechopen.110599*

to *Chlamydia*. Animals that are infected but have not had an abortion do not fully develop immunity to this agent, so they spread the agent to their surroundings during other breeding seasons and lambing periods. Sheep flocks can always be infected with *Chlamydia*, but the most vulnerable period for them to the disease is during lambing [16]. *Clamydia* can survive for weeks or months at low temperatures but has a shorter lifespan at higher temperatures and in harsher environments. There is no evidence that sheep can get infected through sexual transmission [9, 13]. However, if a sheep encounters infection during mating season, the resulting low-weight live offspring or abortion during lambing season suggests sexual transmission. Experimental studies have shown that infection with *C. abortus* did not result in abortion when sheep were artificially inseminated with infected semen or mated with infected rams. However, the infection was still detected in the flock even though it did not result in abortion [15].

Male animals are thought to contribute more to the spread of the disease, as they are not always housed with female animals. Experiments have shown that an infected ram can infect a healthy ewe and her offspring [16]. The presence of *C. abortus* has been detected in the semen of infected 12-month-old rams. When *C. abortus* enters a flock, the first symptoms are not necessarily abortions; general discomfort and discharge from the genital organs within 48 hours are more common. Later on, abortions 2–3 weeks before normal birth indicate that the flock is infected [17]. The waste material is either normal in appearance or has swelling in the umbilical area. The placental membranes have thickened and become reddened. Seven to 10 days after the waste is produced, the infected ewe may have a dirty pink vaginal fluid, which spreads the disease. If the agent remains in the birth canal, it can lead to general discomfort and metritis. Secondary infections result in death. In goats and cattle, vaginitis, endometritis, and placental retention are most commonly seen [15].

The fleece of a lamb that has been aborted may be discolored with a pink-brown substance from placental secretions. In addition to abortion, premature or full-term delivery of stillborn, sickly, or weak lambs can also occur, and most weak lambs do not survive more than 48 hours, even with nursing. Some infected ewes may give birth to healthy lambs, and it is not uncommon for an infected ewe to deliver both a dead and a weak or healthy lamb [16]. The normal pattern of infection in a previously unaffected flock is that a small number of abortions occur in the first year, usually due to the addition of infected replacement female sheep, followed by a sudden increase in abortions in the second year, affecting 30% or more of the female sheep population [7, 8].

The pattern of infection in a previously unaffected flock usually begins with a small number of abortions in the first year, caused by the addition of infected replacement female sheep. In the second year, there is a sudden increase in abortions affecting 30% or more of the female sheep population. Then, in the third year, there is a final enzootic phase in which younger ewes are mainly affected and are generally in their second year of lambing after becoming infected in their first [16]. If adequate control measures are not introduced, the annual incidence of abortion is likely to be around 5–10% [7]. In flocks with an extended lambing season, this pattern may be different, as infected ewes can infect other pregnant ewes later in the same season. In general, the current pregnancy is not at risk unless more than 3–4 weeks remain until lambing [15].

It is believed that the primary infection in sheep is initially established in the tonsils and then spreads to other organs via blood or lymph. In non-pregnant animals, a latent infection is formed, likely in lymphoid tissue, through a process that is influenced by cytokines, especially the pro-inflammatory cytokine interferongamma (IFN-g). IFN-g produced in response to infection with *C. abortus* restricts the organism's growth in living organisms and in the laboratory by inducing the enzyme indolamine 2,3-dioxygenase, which breaks down tryptophan. The *Chlamydia* static effect can be reversed by adding exogenous l-tryptophan [16].

Latently infected sheep do not show any signs of having chlamydial organisms [16]. During pregnancy, the immune system may become weakened, allowing the organisms to multiply and cause a low-grade chlamydaemia, leading to placental infection. In sheep, the placenta is structured with cotyledons, is non-deciduous, and has a syndesmo-epitheliochorial design [16]. Around day 60 of gestation, the mother develops haematomas at the interface between the mother and fetus in the hilus of each placentome these haematomas are believed to allow the Chlamydiae to come into contact with the chorionic epithelium and infect the fetus. No significant changes occur until after day 90 of gestation [13]. The Chlamydiae invade the fetal trophoblastic cells in the hilus of the cotyledon and replicate, causing visible cytoplasmic chlamydial inclusions [8, 13]. Once the infection has taken hold in the trophoblastic cells in the hilus of several placentomes, it spreads to the peri-placentome and intercotyledonary regions of the chorion, causing inflammation, edema, and damage to the epithelial cells and resulting in red, thickened placental membranes. Not all placentomes will become infected, and the degree of inflammation and necrotic damage to the cotyledons and intercotyledonary membrane can vary. In the fetus, the primary pathological changes occur in the liver and can also occur in the lung, spleen, brain, and lymph nodes, although less frequently [16].

The mechanisms behind abortions in sheep infected with *Chlamydia abortus* are not well understood. However, it is believed that the destruction of the chorionic epithelium and related damage may be the cause. A partial impairment of the placentomes can affect the transfer of oxygen and nutrients between the mother and fetus, leading to fetal death [13]. Examination of the infected placental tissues shows a mixture of inflammatory cells, with inflammation and blood clots in the intercotyledonary membranes. The infected tissues also have high levels of TNFalpha, which is not present in normal ovine placentas. This TNF-alpha is believed to cause damage to the placenta and contribute to abortion or premature birth. In vitro studies have shown that the infected trophoblast cells produce PGE2, which may be induced by chlamydial lipopolysaccharide (LPS) through TNF-alpha. The levels of hormones like progesterone, 17b-estradiol, and PGE2 change during chlamydial infection of the placenta and may also play a role in triggering premature labor [13].

#### *5.1.1 Effects on people*

Compared to the human infections caused by *C. psittaci*, which is more commonly found in birds, infection with *C. abortus* is relatively uncommon. A study of 1157 cases of human chlamydiosis in Scotland from 1967 to 1987 showed that only 11 cases were linked to sheep and cattle, while 94 cases were linked to birds [1]. However, a survey of antibody levels in an area in northwest England found no significant difference between the frequency of chlamydial infections in adults working in sheep farming and those in other types of farming or non-farming [8]. Nevertheless, there have been reports of respiratory illness in laboratory staff and workers in vaccine plants and abattoirs, indicating a potential risk for those working in such environments [7, 13].

*Chlamydias as a Zooonosis and Antibiotic Resistance in Chlamydiae DOI: http://dx.doi.org/10.5772/intechopen.110599*

Pregnancy poses the greatest risk of human infection due to the ability of *C. abortus* to settle in the human placenta. Cases of transmission have been reported in various countries, including the United Kingdom, France, the Netherlands and the United States. Although the number of cases each year is low, the potential danger to the pregnant woman and her unborn baby is significant. In the first trimester of pregnancy, human infection will likely lead to spontaneous abortion, while later infections can result in stillbirths or premature labor. These conditions usually follow several days of flu-like symptoms. Pregnant women with the infection may also experience kidney failure, liver problems, and a spreading blood clotting disorder, which can be fatal. Diagnosing *C. abortus* infection can be done through tests such as cell culture or PCR of swabs and fetal samples [7, 8].

Humans can experience abortion caused by *C. abortus* due to exposure to infected sheep or goats, usually transmitted through oral contact, such as handling infected animals or contaminated clothing. Other causes include contaminated food, smoking with unwashed hands, or mouth-to-mouth resuscitation of weak lambs, as well as inhaling contaminated air, for instance from infected lambs placed in front of fan heaters [13].

#### *5.1.2 Diagnosis*

The early and correct identification of the reason for an abortion during pregnancy is essential to implement appropriate measures to limit or stop the spread of the infection. If an abortion occurs in the final two to three weeks of pregnancy and is accompanied by inflamed and necrotic placental membranes, it suggests a potential chlamydial infection, although other microorganisms such as *Coxiella burnetii*, *Campylobacter fetus* ssp. *fetus*, and *Toxoplasma gondii* may also lead to placental damage [7, 8].

Placentae and dead lambs should be immediately sent to a national veterinary laboratory for examination [13]. The process involves placing the samples into strong plastic bags with the use of disposable gloves. The placentae are examined for necrotic placentitis, and stained smears of infected cotyledons are analyzed under a microscope for chlamydial organisms. In case of a possible delay, a piece of affected placental tissue containing an infected cotyledon should be removed and placed in a *Chlamydia* transport medium called SPG. This medium contains 10% fetal calf serum and antibiotics such as streptomycin and gentamicin, but not penicillin. If the placenta is not available, swabs should be taken from the vagina and the moist coats of the lambs to detect any organisms. Suitable staining procedures include Macchiavello, Giemsa, or MZN, with MZN being the preferred choice for routine use. Positive MZN staining, under high-power microscopy, should reveal many small, round, coccoid EBs, either individually or in clusters, stained red against a blue background of cellular debris. Under dark field illumination, the organisms will appear as bright, pale green, round objects [7, 8].

*C. abortus* can be grown from samples of infected tissues, such as eggs from infected hens, endocervical swabs, intercervical membranes, fetal liver, vaginal tampon samples, embryonic eggs of laying hens, and cell cultures. It is important to avoid gut contamination, as there is evidence of enteric *C. abortus* non-pathogenic or commensal strains in ovine infections, and another chlamydial species, *C. pecorum*, is frequently found in feces. Tissue samples or tampons should not be preserved or dried in SPG. *C. abortus* can be isolated from many cell types, but the most commonly used are McCoy, L929, and baby hamster kidney cells [6, 7]. The growth of *C. abortus* in cell culture can

be improved by the presence of cycloheximide in the infection inoculum, pre-treatment of cells with emetine, or exposure of cells to 5-iodo-2-deoxyuridine. After growth, Chlamydiae can be detected by staining smears made from infected yolk sac membranes with Giemsa or staining growing cells in culture with MZN or Giemsa [8, 10].

#### *5.1.3 Control and prevention*

It is necessary to isolate sheep immediately after abortion. As the lambing process continues, attention should be paid to promptly identify and isolate all affected sheep, particularly those giving birth to live lambs instead of stillborns. All dead lambs, placentas, and bedding must be properly disposed of, and lambing pens must be cleaned and disinfected in order to reduce the risk of contamination. It is crucial to thoroughly clean one's hands after handling infectious material before caring for other animals [7, 8].

Long-acting oxytetracycline can be given to reduce the severity of OEA infection in pregnant ewes, but it should only be used in exceptional circumstances. The treatment should start as soon as possible after day 95 of gestation, and multiple doses should be given every 2 weeks until lambing. However, it is important to note that this treatment will not eliminate the infection and cannot undo any damage that has already been done to the placenta. Controlling OEA through proper flock management and vaccination is more effective than relying solely on antibiotics [13].

Pregnant women are recommended to avoid working with sheep, particularly during the lambing period, and to keep away from any potential sources of infection, such as contaminated work clothing. People with compromised immune systems should also exercise caution to avoid contact with sources of infection during the lambing season [7, 8]. Basic hygiene practices like washing hands before eating, drinking, or smoking, and using disinfectants are general steps to be taken but should be strictly followed if an infection is suspected. Disposable gloves should always be used when handling placentas, and under no circumstances should mouth-to-mouth resuscitation be performed on a lamb. Early diagnosis is crucial in treating the infection, which is responsive to antibiotics like tetracyclines and erythromycin when given early [13].

#### **5.2 Avian Chlamydiosis (Psittacosis, Ornithosis)**

*Chlamydophila psittaci* infections can be found all over the world and the bacteria has been found in a wide range of birds, both wild and domesticated, including psittacine birds such as parrots and macaws, game birds, seabirds, garden birds, pigeons, and poultry [14]. These infections are most commonly found in psittacine birds, and the disease is referred to as psittacosis. In other bird species, the same disease is referred to as ornithosis. However, since the disease is similar in all bird species, the term avian chlamydiosis can be used to describe all bird infections caused by *Chlamydophila psittaci* [8, 9].

Studies by Schwartz and Fraser (1982), Bracewell and Bevan (1986), Grimes and Clark (1986), Dorrestein and Wiegman (1989), and Vanrompay et al. (1992) show that the highest incidence of *C. psittaci* infection occurs in psittacine birds. Meanwhile, according to research by Panigrahy et al. (1982), Chiba (1984), Bracewell and Bevan (1986), and Alexander et al. (1989), pigeons have the highest rates of *C. psittaci* infection [13]. Additionally, several economically significant outbreaks of chlamydiosis have been reported in turkeys, and wild birds. Human infections have also been linked to some of these outbreaks [9, 14].

#### *Chlamydias as a Zooonosis and Antibiotic Resistance in Chlamydiae DOI: http://dx.doi.org/10.5772/intechopen.110599*

The spread of *C. psittaci*, a type of microorganism, occurs mostly among birds through breathing in dried-up excrement and secretions, including both eye and nose, from infected birds or by ingesting contaminated feces [13, 14]. The parent birds that are shedding the organism can also pass the infection to their young in the nest, and there is evidence that it can also be transmitted through eggs [14]. *C. psittaci* can also be spread from bird to bird through blood-sucking ectoparasites like lice, mites, and flies, or less frequently through bites or wounds. To avoid the spread of infection, contact between wild birds and poultry should be prevented, as wild birds can act as a potential source of infection [7–8].

Exposure to *C. psittaci* can lead to a variety of infections with varying symptoms, depending on factors like the bird species, virulence, and bird health and stress. The onset of symptoms can range from 3 days to several weeks. In turkeys, the organism can be detected within 48 hours, but symptoms may not appear until 5–10 days later. While subclinical infections may not display symptoms, infected birds can act as carriers and intermittently shed the organism. Acute avian chlamydiosis is a serious infection that affects all major organs, causing symptoms like respiratory distress, lethargy, reduced appetite, ruffled feathers, diarrhea, and discharges from the eyes and nose. The mortality rate from this infection can vary greatly [13].

In summary, birds belonging to the psittacine species can display several clinical signs when infected with *Chlamydia*, such as anorexia, diarrhea, respiratory issues, sinusitis, conjunctivitis, yellow droppings, and central nervous system disturbances [7, 9]. Pigeons can exhibit anorexia, diarrhea, conjunctivitis, swollen eyelids, and rhinitis in acute cases and lameness, torticollis, opisthotonus, tremor, and convulsions in chronic cases [13, 14]. Turkeys infected with highly virulent serovar D strains can experience severe symptoms such as anorexia, cachexia, yellow-green diarrhea, low egg production, conjunctivitis, sinusitis, and sneezing, with mortality rates ranging from 10 to 30% [15]. If turkeys are infected with a virulent B serovar instead of a serovar D, the infection will run a milder course. Symptoms such as mild anorexia and diarrhea may be noticeable [7, 15]. Ducks can show symptoms such as trembling, unsteady gait, conjunctivitis, nasal discharge, and depression. Chickens tend to be relatively resistant to *Chlamydia*, but in some cases, they may develop blindness, weight loss, and an increase in mortality rate [13].

#### *5.2.1 Effects on people*

Psittacosis or ornithosis is a disease that can be transmitted from birds to humans. The largest group affected by this disease are bird fanciers and pet bird owners, as well as people whose jobs put them at risk of exposure such as pet shop employees, aviary workers, veterinarians, laboratory workers, poultry processing plant employees, farmers, and zoo workers [13]. From 1996 to 2001, there were reports of 1620 cases of human psittacosis in the United Kingdom, 661 in Germany, and 165 in the United States. These figures are believed to underestimate the actual number of cases, as psittacosis can be difficult to diagnose. The disease is considered to be an occupational hazard and is recognized as a prescribed disease for Industrial Injuries Disablement Benefit in the United Kingdom [15]. The OIE Central Bureau maintains a world database of animal diseases and zoonoses, which can be accessed through the Handistatus web interface [13].

Infection with *C. psittaci* can occur through inhaling dried feces or respiratory secretions from infected birds, or by having direct contact with their feathers, tissues, or secretions, including through mouth-to-beak contact or open skin wounds. Some virus strains are very contagious to humans, and even a short exposure could lead to infection. Although there have been suggestions for person-to-person transmission, it is not considered a common occurrence [7, 8].

The human disease known as psittacosis has an incubation period that is generally estimated to be between 5 to 14 days, but in some instances, it may last up to a month. Symptoms of psittacosis can be either mild, with flu-like symptoms such as fever, headache, joint and muscle pain, photophobia and sore throat, or severe, with symptoms of atypical pneumonia including a non-productive cough and difficulty breathing [14]. During the acute phase of the illness, the white blood cell count is often normal, although a decrease in white blood cells, known as leucopenia, can occur in approximately 25% of cases. The pulse rate is typically slow relative to the elevated body temperature, and a rash may also be present. Chest X-rays often reveal signs of pneumonia [14, 15].

In addition to affecting the respiratory system, psittacosis can also result in complications in other organs, such as myocarditis, endocarditis, hepatitis, encephalitis, meningitis, and kidney and neurological issues [7, 8]. Those most at risk of developing these complications are the very young, the elderly, and individuals with weakened immune systems [13].

In order to diagnose chlamydiosis in birds, it is important to detect the presence of the *Chlamydia* organism or antibodies to the infection. Symptoms such as an increased white blood cell count, changes in liver enzyme activity, radiographic signs of liver and spleen enlargement, and air sac inflammation may indicate the presence of chlamydiosis. However, a definitive diagnosis requires the detection of the organism or the presence of antibodies. The tests used to diagnose avian chlamydiosis are similar to those used to diagnose other chlamydiosis [15].

The most effective methods for detecting antigens involve the use of PCR technology. There are several PCR tests mentioned in the scientific literature that target different genes such as ompA (MOMP), pmp (or pomp) genes, and 16S and 23S rRNA [15]. These tests have been successfully used on bird samples, but more research is needed to fully validate their use for diagnosing psittacosis. These PCR tests and the MIF test can be used to differentiate infections caused by the bacterium *C. psittaci* [13, 15].

The methods for diagnosing human psittacosis are similar to those for birds. The organism may be cultured from bodily fluids such as sputum, blood, pleural fluid, or biopsy material, but this is not typically done. Instead, infection is usually diagnosed based on a serological analysis of paired sera taken at least 2 weeks apart. Chest X-rays can also help with the diagnosis and may show signs of lobar, patchy, or interstitial infiltrates. MIF assays and several PCR tests can be used to differentiate *C. psittaci* infections from other chlamydial infections like *C. pneumoniae* and *C. trachomatis* in human patients [15].

#### *5.2.2 Control and prevention*

In short, controlling human psittacosis requires controlling the disease in birds. This can be done by maintaining high standards of aviary husbandry, including daily cleaning and disinfection of cages with effective disinfectants such as 70% isopropyl alcohol, 1% lysol, or a 1 in 100 dilutions of household bleach. All waste feed and litter must be disposed of as they can remain infectious for several months. Minimizing aerosols can be done by using litter that does not produce dust, spraying floors with disinfectant or water, and providing sufficient exhaust ventilation [13].

#### *Chlamydias as a Zooonosis and Antibiotic Resistance in Chlamydiae DOI: http://dx.doi.org/10.5772/intechopen.110599*

These measures are aimed at reducing the risk of infection with avian influenza or bird flu, which is a highly contagious virus that can cause severe disease in birds and in some cases, can be transmitted to humans [7, 8]. The use of protective clothing and respirators helps to prevent airborne transmission of the virus, while the use of biological safety cabinets and local exhaust ventilation helps to minimize the risk of exposure to the virus through contaminated surfaces and respiratory droplets. The use of detergent and water when performing necropsies helps to reduce the spread of the virus through aerosols, while heat-treating birds and providing respiratory protection in poultry processing plants helps to reduce the risk of exposure to the virus for workers handling infected birds [8, 14].

Treatment for avian chlamydiosis typically involves the use of tetracycline drugs, as recommended by Vanrompay et al. and the Centers for Disease Control [13]. This treatment involves the administration of CTC through medicated feed, which is commercially available as pellets for larger birds and millet for smaller ones. However, it is important to monitor the bird's food consumption, as acceptance of the medicated feed may be variable. The diet should not have a calcium content higher than 0.07% as it interferes with the uptake of CTC. Most companion birds should receive the medicated diet for a minimum of 45 days, while poultry should receive it for a minimum of two weeks, but it should be discontinued two days before slaughter. In cases where the bird is severely ill, oral or parenteral treatment may be necessary, with doxycycline being the preferred drug for oral treatment with a recommended dosage of 25 to 50 mg/kg once a day on an empty crop to aid absorption [14]. Injectable formulations of doxycycline or oxytetracycline may be given intramuscularly, but the latter should only be given to stabilize severely ill birds as it can cause tissue necrosis. Enrofloxacin, a quinolone approved for use in domestic animals, is also being evaluated for the treatment of avian chlamydiosis. During treatment, supportive care such as intravenous fluid therapy and a heated, uncrowded environment may be necessary. It is important to eliminate the possibility of reinfection by thoroughly cleaning and disinfecting the environment after recovery [13].

Currently, there are no commercially available vaccines for avian chlamydiosis. However, research has been done in the past, and some promising results have been seen. In the 1970s, a vaccine was created using an inactivated form of the bacteria that causes chlamydiosis, *Chlamydia psittaci*, which successfully induced a cell-mediated immune response and protected 90% of turkeys against the disease after being challenged [15]. More recently, another vaccine was developed using DNA from *Chlamydia psittaci*, which has resulted in a significant level of protection in turkeys, generating both humoral and cell-mediated responses [13].

#### **5.3 Other Animal Chlamydioses**

In simpler terms, *Chlamydia psittaci* and *Chlamydia abortus* are both known to cause human illnesses, but *Chlamydia felis* is the only other type of *Chlamydia* that has been linked to human infections. This type of *Chlamydia* is commonly found in cats and can cause eye infections, especially in young kittens [7, 8].

In simpler terms, there have been several reports of people getting sick from being in close contact with cats, including eye infections, abnormal liver function, heart and kidney problems, and atypical pneumonia [4]. A recent study showed that a patient with a chronic eye infection and one of the patient's cats had the same type of *Chlamydia* (*C. felis*), which suggests that the infection was transmitted from the cat

to the person. However, such infections are not commonly reported, which could be because they are rare or because they are not properly diagnosed [13].

Although we have some knowledge about the potential of *C. abortus*, *C. psittaci*, and *C. felis* to cause disease in humans, we do not know much about the disease-causing potential of other chlamydial agents in humans [13]. *C. pecorum*, which causes significant diseases in animals, affects many domestic farm animals such as sheep, goats, cows, horses, and pigs, causing pneumonia, conjunctivitis, polyarthritis ("stiff lamb disease"), inapparent intestinal infections, mastitis, metritis, and encephalomyelitis [8]. However, no zoonotic cases have been reported so far. *C. suis* in pigs causes conjunctivitis, enteritis, and pneumonia. *C. pneumoniae*, which causes disease in humans' respiratory tract, has also been isolated from frogs, koalas, and horses [7, 8]. It has been associated with chronic heart disease, Alzheimer's disease, reactive arthritis (Reiter's syndrome), and asthma [7]. Waddlia chondrophila, recently isolated from aborted bovine fetuses, belongs to the Chlamydiales order, and more data is needed to determine its zoonotic potential [13].


UTI Caused by *Staphylococcus saprophyticus*

*Benyamin Djawadi, Nazila Heidari and Mojtaba Mohseni*

#### **Abstract**

Coagulase-negative Staphylococci (CoNS) are one of the most frequently isolated bacteria in the clinical microbiology laboratory. These bacteria are normal inhabitants of human skin and mucous membranes; also, they have emerged as significant nosocomial pathogens. *Staphylococcus saprophyticus* is a Gram-positive bacterium well known for causing uncomplicated urinary tract infections in young sexually active females, responsible for complications including urinary tract infections, epididymitis, prostatitis, and acute pyelonephritis. CoNS can be divided into two groups based on susceptibility to novobiocin. The novobiocin-susceptible species include *S. epidermidis*, *S. haemolyticus*, *S. hominins*, *S. lugdunensis*, *S. schleiferi*, and the novobiocin-resistance species, including *S. saprophyticus* and *S. xylosus*. The acute uncomplicated UTI, including cystitis and pyelonephritis, is frequent in an immunocompetent nonpregnant female population, the second most common cause of community-acquired urinary tract bacterial infection in women after the *Escherichia coli*. *S. saprophyticus* is a part of the normal human flora which colonizes the rectum, urethra, cervix, and gastrointestinal tract. Bacterial colonization of the bladder and ureter epithelium occurs via several types of adhesin, including hemagglutinins with autolytic properties. Also, some strains can create and produce biofilms to increase their pathogenicity.

**Keywords:** *Staphylococcus saprophyticus*, UTI, coagulase-negative staphylococci, *S. saprophyticus*, CoNS, urinary tract infections

#### **1. Introduction**

Staphylococcus organisms can be coagulase-positive or negative, and Coagulasenegative Staphylococcus microorganisms are associated with human infections. *S. saprophyticus* is a clustering Gram-positive Commensal CoNS (Coagulase-negative Staphylococci), non-motile, non-spore, non-hemolytic coccus and Novobiocinresistant which is a common urinary tract pathogen accounting for 10–20% of cases in young women, rarely associated with urinary tract infections in male populations, with the cell membrane that's made up of the typical lipid-protein bilayer made of phospholipids and the cell wall which is composed of peptidoglycan and teichoic acid that function to maintain the shape of the cell associated with adhesions, unique adhesion protein which allows it to attach to human uroepithelial cells and abundant

transporter systems to adapt to changing pH and osmolarity [1]. *S. saprophyticus* is a common cause of urinary tract infections in the young, sexually active female population. There are multiple chances of developing prostatitis and urethritis in immunocompetent hosts. *S. saprophyticus* can be differentiated from other coagulasenegative Staphylococcus by its resistance to Novobiocin, and like other uropathogens, *S. saprophyticus* can produce ammonia by utilizing urease [2]. Coagulase-negative Staphylococcus can be found on the skin, rectum, urethra, and cervix as a microbiome [3–5]. *S. saprophyticus* is the second most common cause of UTI after *E. coli*. Over 40% of all sexually active females contain *S. saprophyticus* as part of their normal genitourinary flora [3–5].  This bacterium can encode different transport proteins, enabling it to adjust to environmental pH changes and proliferate urine.

#### **2. Definition of UTI**

Urinary tract infection, also called UTI, is one of the most common bacterial infections; it is characterized into different groups: young women with uncomplicated cystitis, female with recurrent cystitis, women with acute uncomplicated pyelonephritis, asymptomatic bacteriuria, and adults with cystitis complications. A complicated UTI is a condition that increases the risk of developing severe complications like treatment failure (**Figure 1**) [7].

UTI is a term to explain any urinary tract infection that concludes with asymptomatic bacteriuria, pyelonephritis, and cystitis. Acute complicated UTIs are typically between 27 to 44% of the healthy female with normal urinary tracts. The incidence of symptomatic UTI in men younger than 50 years is much lower than in women [8]. Asymptomatic bacteriuria is found in 5% of young women and rarely in men younger than 50 [9]. Most uncomplicated UTIs in the healthy female population has resulted from uropathogenic, found in rectal flora in men; colonizing uropathogens may also come from the partner's vagina or rectum [10]. Factors predisposing patients to develop complicated UTIs are generally caused by urine flow which facilitates the

**Figure 1.**

*Classification of UTIs in young women.*

**Table 1.**

*Risk factors for common UTIs.*

entry of uropathogenic into the urinary tract by bypassing the host defense system [11]. Recurrent types of cystitis in healthy female is triggered when an infection is repeated during different period, which is caused by the persistence of the infected strain in the fecal flora [12]. Asymptomatic bacteriuria occurs in patients without any urinary infections [13]. Both pyelonephritis and cystitis are UTIs involving the bladder and kidneys; based on the risk factors such as gender and having sexual intercourse, other complications can be classified into complicated or uncomplicated UTIs [14]. These classifications are made to help patients in need of diagnostic tests or even more extended periods of antimicrobial treatments. On the other hand, an uncomplicated UTI can occur in a healthy nonpregnant woman with beneficial flora and no history of bladder compilations such as bladder catheterization [15]. Diabetic women are classified as a group having complicated infections. As mentioned before, cystitis and pyelonephritis are depended on the patient's risk factors, such as UTI history, diabetes, age, e, and gender. Although cystitis can progress to hostile stages and cause acute pyelonephritis, the occurrence is rare [16]. Acute pyelonephritis includes sepsis, kidney infection, and perinephric abscess [17]. Recurrent UTIs are commonly defined as infections within 12 months or predisposition to two disorders within 6 months [18]. Females diagnosed with UTI will develop a recurrence within 3 months [19]. A summary of risk factors for common UTIs is shown in **Table 1** [20–23].

For different reasons, postmenopausal women with the risk of developing UTIs, such as lower levels of systemic estrogen, play essential roles in adjusting the vaginal pH and maintaining the normal vaginal flora. The lower estrogen level in postmenopausal women is a crucial factor contributing to UTIs in the community. Based on a Cochrane review, vaginal estrogen, cranberries, Hyaluronic acid, and Chondroitin sulfate have a big part in decreasing recurrent UTIs [24]. Probiotics are thought to contribute to preventing UTIs by restoring the normal vaginal microbiome [25].

#### **3. Pathogenicity and virulence factors**

In the 1990s, Kloos and Bannerman [26] shed light on the clinical importance of CoNS. Today, CoNS represent one of the most virulent nosocomial pathogens which had a crucial impact on human health [27]. *Staphylococci* colonize and develop complications by owning the strain-specific virulence strategies for the aggression, invasion, and adherence factors essential for pathogenicity and evading the host immunity mechanisms. Once the periurethral region contaminated with *S. saprophyticus* from the gut, the microorganisms will colonize the urethra before traveling

up to the bladder. To attach to the uroepithelial cells, *S. saprophyticus* expresses adhesins: extracellular slime and fibronectin binding proteins [27]. *S. saprophyticus strains can also produce urease which hydrolyzes the ammonia to ammonia and carbon dioxide, ammonia will support the bacteria's survival with the pH increasing* [28]*.* As soon as the host immune system cells sense the bacterial infection, phagocytes, macrophages and neutrophils are being called to engulf the bacteria, then the bacteria are trapped inside a phagosome, which occurs when the phagosome fuses with a specialized vesicle that has degradative agents. However, *S. saprophyticus* bacteria can eventually escape the digestion pocket in coating themselves with the hosts proteins in order to disguise themselves to prevent from being identified by antibodies or blood proteins [27].

#### **3.1 Adherence to surface and biofilm formation**

Biofilm production has a significant role in infection. It's an accumulation of microorganisms (primarily endogenous) and their self-developed polymeric matrix extracellular products which form on the surface. (e.g., the ureteral stents) The first moment of *Staphylococci* colony formation is adherence to host epithelium cells which increases its virulence and also its antibiotic tolerance in comparison with isolates without biofilm production. Adherence is critical in initiating UTI pathogenesis [28]. The colonization of the multilayered biofilm is considered an essential step in the pathogenesis of a foreign host [29]. In the biofilm, bacterial cell agglomerates, extracellular DNA (eDNA), pili, flagella, and extracellular polymeric substances are encased in an extracellular material composed of teichoic acids to protect the microorganism from the host immune system [30]. After insertion into the host, ions, polysaccharides, and other components diffuse toward the host cells and provide a receptor site for bacterial adhesins, enhancing the adherence rate. In the next step, bacterial cells release protons as signaling molecules to develop a concentration on the surface, which can lead to the formation of a biofilm format. Members of *Staphylococcus* produce various adhesins, such as proteinaceous ones, grouped into surface-anchored proteins, also termed cell wall-anchored (CWA) proteins [31].

In many developing countries the UTIs caused by *S. saprophyticus* and the structure of biofilm are not that well studied but the phases of biofilm formation by CoNS can be divided into initial attachment, Accumulation, Maturation, Detachment, and dispersal. The initial extension of CoNS is followed by the accumulation phase, which includes cell proliferation, intracellular adhesion, and maturation. A developed biofilm is built up of three layers: linking film, which cattachtch to the surface of the tissue, forming a base layer of compact microorganisms, and surface film, where free-floating bacterial cells are spread over the surface [32]. Some *Staphylococci* strains have been associated with a 240-kDa protein called biofilm-associated protein (Bap), which mediates the attachment and biofilm accumulation and plays a role in developing bacterial inflammation, recurrent cystitis, and chronic prostatitis [33]. The surface virulence factors include proteins such as Aas (Binds to the fibronectin), UafA (Binds to bladder epithelia cells), UafB (Binds to fibronectin and bladder epithelial cells), Srdl (Binds to collagen) [32]. The chosen antibiotic in uncomplicated *S. saprophyticus* UTIs is nitrofurantoin 100 mg orally twice daily for five to seven days in more complicated cases [30]. Also the prescribing of Trimethoprimsulfamethoxazole (TMP-SMX) may be given alternatively in uncomplicated cases [30]. The general steps of biofilm formation in *S. saprophyticus* [31].

#### **3.2 Teichoic acids**

Teichoic acids are involved in the adherence of *S. saprophyticus* to host epithelium cells, which dose-dependently is promoted by the attachment of these microorganisms, which act as a bridge between fibronectin polymer material and bacteria [34]. Teichoic acids have been shown to exert binding functions for matrix molecules in Gram-positive, including *Staphylococci*. Wall teichoic acid (WTA) is linked to the peptidoglycan (PG), composed of glycerol phosphate units, substituted at the positions of glycerol with alpha-glu-cocaine, alpha-glucose, alpha-6-D-alanyl-glucose [35].

#### **3.3 Aggressive virulence factor**

Coagulase-positive staphylococci have been isolated from more than 95% of cases of toxic shock syndrome (TSS). Toxic shock syndrome toxin-1 (TSST-1) is produced in more than 92% of cases. Compared *to S. aureus*, CoNS have much lower levels of aggressiveness and mostly are not producers of super toxin antigens. The capability of CoNS to produce exfoliate toxins has been doubted since the late 1980s [36]. TSST-1 producing coagulase-negative staphylococci were also isolated from different cases, but the illness episodes were mild recurrences of TSS since they did not meet all the TSS criteria. The strains of presumed positive toxic shock syndrome toxin 1 (TSST-1) cannot produce and exert TSST-1 [37].

#### **3.4 Production of Lantibiotics**

Lantibiotics are a group of post-translationally modified peptides that contain unusual amino acids such as lanthionine residues. Commensal staphylococci produce Lantibiotics like Nisin A (act against a wide variety of bacteria, including strains of Lactococcus, Streptococcus, and Staphylococcus), Epidermin, and Mersacidin [38]. These groups of bacteriocins are classified into cationic antimicrobial peptides (CAMPs), which are classified into two types, including Type A (e.g. Nisin, Bisin, Subtilisin, and Epidermin) and Type B (e.g. Mersacidin, Duraycin, and Cinnamycin). The production plays a role in bacterial interference, creating an ecological niche for *Staphylococci* [39]. These antibiotic peptides also include methyl xanthine amino acids, which can bind to bacterial cell wall precursor lipid components and disrupt cell wall production. Type A kills rapidly by pore-forming mechanisms, and Type B inhibits peptidoglycan biosynthesis [40].

### **4. Identification and grouping of CoNS by novobiocin testing**

Novobiocin is an antibiotic that interferes with DNA replication and binds to DNA gyrase, leading to the blocking of adenosine triphosphates (ATPase) activity. If a CoNS isolate is recovered from urinary tract specimens, testing for resistance to novobiocin will distinguish *S. saprophyticus* from other clinical CoNS [41]. This method is based on the disc diffusion test using 5-μg novobiocin discs on Hinton agar or blood agar medium [41]. As the microorganism multiplies during incubation to produce a surface of confluent growth, cells are exposed to the antibiotic that diffuses into the agar from the paper disk. If the bacteria are susceptible to novobiocin, there will be a formation of a zone of inhibition around the disk representing an area where the bacterial growth has been prevented [42]. A zone of inhibition less than 16 mm is indicative of novobiocin

resistance. The CoNS have been classified into two groups based on this particular pattern. The ones demonstrating novobiocin susceptibility include *epidermidis, S. capitis, S. haemolyticus, S. hominis, S. lugdunensis, S. warneri* and *S. saccharolyticus.* The novobiocin-resistant group consists of: *S. kloosii, S. saprophyticus,* and *S. xylosus* [42].

#### **5.  Regarding antimicrobial resistance, CoNS have been divided into two groups: 1-Susceptible to antimicrobial agents and 2-those exposed to antibiotics in the hospital. CoNS are thought to show an exquisite reservoir of genetic elements that led to resistance to β-lactam antibiotics and other antimicrobial classes [43]. In *Staphylococci*, including CoNS species, penicillin-binding protein (PBP) expression can lead to β-lactam resistance, such as cephalosporins and carbapenem [44].

#### **6. Preventing recurrent UTI/probiotics**

Nonantimicrobial and antimicrobial prophylaxis regimens have been studied in the female population with recurrent UTIs. Probiotics can prevent UTIs by helping to restore the normal vaginal microbiome. Studies investing in *Lactobacillus* spp. to prevent recurrent UTIs in women have found a link between recurrent symptomatic UTIs and probiotics. Still, additional studies are needed for final decisions to be made [45].

#### **6.1 Preventing recurrent UTI/cranberries**

The mechanism of cranberries preventing UTI is yet to be discovered. Although, studies show that the daily drinking of 300 mL of cranberry juice plays a role in preventing this complication due to the function of proanthocyanidins found in cranberry, which inhibit the microorganisms from adhering to the bladder walls and the epithelium cells and make tissues damages [46].

#### **6.2 Preventing recurrent UTI/hyaluronic acid and chondroitin sulfate**

The glycosaminoglycan layer has been found in the bladder wall, made of Hyaluronic acid and Chondroitin sulfate, which plays a role in preventing the adherence of microorganisms to the urothelial cells [47]. A damaged glycosaminoglycan layer could lead to an increased rate of recurrent UTIs in both the male and female populations (**Table 2**) [48].

#### **7. Treatment and resistance**

Treatment with antibiotics has been indicated in complicated UTIs and pyelonephritis. There is no indication for routine susceptibility tests for UTI due to *S. saprophyticus* because it's usually susceptible to empirical therapy for uncomplicated UTI [49]. The antibiotics for uncomplicated UTI are sulfonamides, ampicillin, and nitrofurantoin 100 mg twice daily for five days or trimethoprim/sulfamethoxazole

#### **Table 2.**

*Prophylaxis for recurrent UTI.*

160/800 twice daily for three days. In acute uncomplicated UTIs, NSAIDs are a preferred analgesic [50, 51]. resistance to trimethoprim/sulfamethoxazole can lead to failure in empirical therapy, so an antimicrobial susceptibility test is necessary in these cases [52]. Baicalin can be used in UTI treatment because of its inhibitory effects on more efflux gene expression, which encode the msrA efflux pump, which is essential in multidrug resistance in staphylococcus strains [52]. Nalidixic acid has no rule in treating *S. saprophyticus* UTI due to bacterial resistance. In some studies, the terms methicillin resistance and Oxacillin resistance is being used and recording to CLSI guideline the cefoxitin disc is also being used [54]. Oxacillin resistance occurs due to the production of pB2a in the microbe [52]. Pivmecillinam is another acceptable antibiotic in pregnant patients, while mecillinam is not practical [54] (**Table 3**).

A single dose of fosfomycin trometamol also is a good choice in treating uncomplicated UTI and pregnant patients with lower UTI as a first-line empirical therapy (**Figure 2**) [53].

Some *S. saprophyticus* species are resistant to Cefixime and fosfomycin trometamol naturally, so it does not recommend to use these drugs in treating acute

#### **Table 3.** *Treatment with antibiotics.*

**Figure 2.** *Antibiotic-resistance profile [54].*

pyelonephritis. Ceftriaxone can be used instead of Cefixime and ciprofloxacin for treating acute pyelonephritis [55].

#### **8. Conclusion**

CoNS are versatile Gram-positive bacteria, either coagulase-positive or coagulasenegative, with mucous membranes and skin niches. Coagulase-negative organisms should not always be thought of as contaminants in tissue infection. CoNS rarely get hostile because most of them lack virulence factors. Based on the rise of weak and susceptible patients, CoNS consequently have become major nosocomial pathogens, and their virulence functions have led to complications such as urinary tract infections (UTI). Urinary tract infections are among the most frequent types of hospital and community infections especially in women than man because of their anatomical differences. Pyelonephritis and cystitis are the most common UTIs based on the factors affecting patients and are classified as complicated or uncomplicated. Uncomplicated infections are those in nonpregnant healthy women with normal genitourinary tract and medical history without any recent UTIs. *S. saprophyticus* is one of the major pathogens causing UTIs. It is a Coagulase-negative Staphylococci (CoNS), colonizing the ureter epithelium via adhesins with the ability to produce biofilms to increase their pathogenicity, however little has being discovered about antibiotic resistant patterns and biofilm functions in this topic. We must know that the treatment of UTIs depends on the S. saprophyticus growing resistance to antibiotics. The big part of *S. saprophyticus* infections can be treated with antibiotics as said before but if left untreated, can progress to complications. A great deal of knowledge of antimicrobial resistance patterns is essential when choosing an antibiotic agent. So many questions about the ecology, pathogenesis, and phylogeny of CoNS have not been answered yet, but hopefully, following the development of new methodology tools, further research can be approached.

*UTI Caused by* Staphylococcus saprophyticus *DOI: http://dx.doi.org/10.5772/intechopen.110275*

#### **Author details**

Benyamin Djawadi1 \*, Nazila Heidari<sup>2</sup> and Mojtaba Mohseni3

1 Faculty of Medical Sciences, Parasitology Department, Tarbiat Modares University, Iran

2 Department of Medical Science, Iran University of Medical Sciences, Tehran, Iran

3 Department of Microbiology, University of Mazandaran, Babolsar, Iran

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**Chapter 3**


**5. Conclusions**

In conclusion, these results show the efficacy of trap cropping for the management of the rice RKN phytosanitary problem in most rice-growing areas, especially those with water shortages. In climatic and pedological areas similar to the Lombardy region, the duration of the three trap crop cycles could be just over two months. This technique of decreasing the nematode population density in the soil, therefore, has a much shorter time of action than flooding method (as indicated among the phytosanitary measures reported in Ministerial Decree of 6 July 2017). However, future studies are necessary to establish the most effective number of trap crop cycles that are useful to reduce the presence of *M. graminicola* in the infested soils, maintaining its density below the level that allows the optimal growth of rice plants. Moreover, this technique could be also inserted in integrated pest management programs as a low environmental impact agronomic practice, compared to the flooding method, to control this damaging rice pest.

**Author Contributions:** Conceptualization, S.S., G.T., L.M., G.M., B.C., and M.C.; Methodology, G.T., L.M., and G.M.; Software, G.M.; Validation, G.T., G.M., and L.M.; Formal Analysis, G.M. and G.T.; Investigation, S.S., G.T., L.M., A.F., G.M., B.C., and M.C.; Writing—Original Draft Preparation, G.T., L.M., and G.M.; Writing—Review & Editing, G.T., L.M., G.M., M.C., and S.S.; Supervision, M.C., B.C., and P.F.R.; Funding Acquisition, M.C., B.C., and P.F.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Piersandro and Caterina Rossi, the owners of the Cascina Scalina farm for their kind cooperation and Alessandro Cini for helpful statistical analysis. The authors also wish to thank Kathleen Collins Tostanoski for the English revision.

**Conflicts of Interest:** The authors declare no conflict of interest.