**2. Antibiotic susceptibility and resistance mechanisms in human chlamydial infections**

*Chlamydiae* are obligate intracellular bacteria that have a distinctive bi-phasic life cycle that typically lasts 40–72 hours [3]. The extracellular infectious form is called the elementary body (EB); however, EB does not have metabolic activity and is resistant to antibiotics. On the other hand, the intracellular form, the reticulate body (RB), is the replicative element that synchronously duplicates every 2 to 3 hours and is the target for antibacterial agents [2, 3]. To achieve optimal bacterial clearance, antibiotics with good intracellular penetration and preservation of antibiotic concentration throughout the life-cycle of the organism are needed.

*Chlamydiae* are sensitive to a wide range of antibiotics such as tetracyclines, macrolides, fluoroquinolones, rifamycins, and clindamycin, which prevent deoxyribonucleic acid (DNA) and protein synthesis and have good activity against intracellular bacteria. Beta-lactam antibiotics and especially penicillins may exhibit *in vitro* efficacy but are linked to the persistence of chlamydial infection and therefore are not recommended for treatment. However, studies revealed that amoxicillin may exhibit higher efficacy than erythromycin in pregnant women with chlamydia disease and due to the paucity of antibiotic options in this period amoxicillin might be offered as an alternative therapy during pregnancy [4–6]. Among other antibacterial classes, aminoglycosides and glycopeptides are ineffective against chlamydial infections since *Chlamydiae* are constitutively resistant. Also, trimethoprim is not effective against *Chlamydia* spp. but *C. trachomatis* is susceptible to sulfonamides [7]*.*

Despite having a severely condensed genome of only about 1 megabase, *Chlamydia* spp. have a highly reserved genomic structure and evidence of horizontally acquired foreign DNA is scarce. Antibiotic resistance is not common in human chlamydial infections but significant resistance phenotypes such as heterotypic resistance, which is characterized by few organisms (less than 1%) that are resistant to antibiotics at concentrations higher than their minimal inhibitory concentrations (MIC), might be expressed [8]. Currently, *in vitro* susceptibility testing of *Chlamydia* spp. is not

*Treatment of Chlamydial Infections DOI: http://dx.doi.org/10.5772/intechopen.109648*

methodized because resistant clinical isolates are rare; without host cells, the organism cannot be recovered; the identification and interpretation of antibiotic resistance are not standard; and the impact of heterotypic resistance is uncertain [3].

*In vitro* studies have shown that antibiotic exposure can lead to the accumulation of point mutations, which result in antibiotic resistance in *Chlamydiae.* Stable genetic resistance and transmission of resistance genes across strains have not been reported for human chlamydial infections; however, genetically stable tetracycline resistance has been detected in *Chlamydia suis* (which causes disease in pigs) isolates and acquired tetracycline resistance (Tet<sup>R</sup> ) has been shown *via* horizontal gene transfer and homologous recombination mechanisms [3, 6, 9, 10].

As mentioned above, the mechanisms that might be related to antibiotic resistance in human chlamydial infections are the persistence of the microorganism, heterotypic resistance, and antibiotic point mutations, which will be detailed as follows:

#### **2.1 Chlamydial persistence**

When exposed to stressful circumstances such as beta-lactam antibiotics, interferon-gamma (IFN-γ), a lack of iron supplements, or amino acids, *Chlamydia* spp. exhibit characteristics of chlamydial persistence [3, 11]. Among them, beta-lactam antibiotics constitute a major problem since they are among the groups of antibiotics that are most frequently used for the treatment of various infections [12]. Similar to gram-negative bacteria, *Chlamydia* spp. have an outer membrane but they lack peptidoglycan while having genes that code for proteins necessary for its formation [13]. Studies have shown that *C. pneumoniae* and *C. trachomatis* can synthesize an unusual truncated type of peptidoglycan, which may serve as a target for beta-lactam antibiotics. However, following antibiotic exposure, the pathogen enters a stage known as persistence in which the stressed *Chlamydiae* remain viable but non-culturable. Moreover, it was shown that the persistence of *Chlamydiae* may remain in culture for months and result in clinical treatment failure because of phenotypic resistance to antibiotics, which are often quite efficient [3, 14]. As an example azithromycin, phenotypic resistance emerged in *C. trachomatis* isolates after pre-exposure to penicillin in experimentally infected endometrial epithelial cells *in vitro*, rendering the pathogen refractory to the given medication [15]. Azithromycin and ofloxacin resistance in *C. pneumoniae* and doxycycline resistance in *C. trachomatis* have both been demonstrated to increase in response to environmental changes [16, 17]. More importantly other than phenotypic resistance, recent data indicate that chlamydial persistence may potentially be associated with chronic diseases such as reactive arthritis, chronic prostatitis, asthma, and atherosclerosis [3, 7].

#### **2.2 Heterotypic resistance**

Although many antibiotics are effective against chlamydial infections, antibiotic failure rates that vary between 5 and 23% of cases have been reported in chlamydia disease. However, chlamydial resistance is not frequent in humans. Much of these failures have been connected to re-infection and antibiotic compliance issues [6]. In the literature, antibiotic-resistant, *C. trachomatis* clinical isolates are scarce and displayed characteristics of heterotypic resistance as previously explained [3]. It was hypothesized that heterotypic resistance may be due to the slow growth of the organism in certain environments or the exhibition of an adaptive response that makes the pathogen resistant to antibiotics [9]. On the other hand, resistant isolates exhibited reduced fitness, which impaired long-term survival or led to the loss of resistance pattern upon serial passaging. It is considered that this phenotypic alteration seems to impair the transmission and therefore prevents the emergence of non-susceptible clones [3, 18, 19].

### **2.3 Genes and mutations associated with human chlamydial infections**

For the treatment of chlamydial infections, tetracyclines and macrolides are quite effective. It is commonly acknowledged that antibiotic misuse or overuse increases the chance of microorganisms' developing antibiotic resistance, which poses a critical and dangerous public health issue globally [20, 21]. However, human chlamydial infections have not been associated with persistent and heritable genetic resistance. *In vivo* studies have shown that *Chlamydiae* may acquire resistance through several mutations to antimicrobials [3]. Additionally, it has been demonstrated that the serial passage of *Chlamydia* spp. leads to the selection of resistant isolates if exposed to sub-inhibitory antibiotic doses [21].

Tetracyclines are bacteriostatic antibiotics that prevent aminoacyl tRNAs from binding with ribosomal 30S subunit, hence inhibiting bacterial protein production [3]. Doxycycline is a semisynthetic tetracycline and is recommended as the primary therapeutic option against *C. trachomatis* infections. Apart from human infections tetracyclines are widely used in veterinary medicine. The emergence of genetically stable TetR resistance is first described in the 1990s and observed in *C. suis* isolates recovered from pigs both healthy and sick. Since then, the threat of transmission of TetR resistance to other members of *Chlamydiae* is of great concern [10]. Although *in vitro* tests have shown that TetR may be horizontally transmitted from *C. suis* to clinical isolates of *C. trachomatis* during co-culture; to date, antibiotic failure due to stable TetR has not been reported in human chlamydial infections. In a study by O'Neill et al., a porB gene mutation was discovered in two clinically resistant isolates but the isolates were reported to be phenotypically sensitive to tetracyclines *in vitro* and stable genetic resistance was not evident [22].

Macrolides are a group of antimicrobials that bind to bacterial 50S ribosomal subunit and impair bacterial growth due to the inhibition of protein synthesis. They are also effective front-line classes of antibiotics used for the treatment of chlamydia infections. The size of the macrocyclic lactone ring determines whether the group is classified as having a 12-, 14-, 15-, or 16-membered ring. Among macrolides, erythromycin (14-membered first generation), clarithromycin (14-membered second generation), and azithromycin (15-membered) are widely used for chlamydial infections [23]. The 23S rRNA gene alterations that make antibiotics less able to bind to the 50S ribosomal subunit, which is necessary for bacteriostatic action, usually cause macrolide resistance. It has been demonstrated that mutations to the 23S rRNA gene can cause *C. trachomatis* and *C. psittaci* to become resistant to macrolides. In addition by means of alterations in the rplD and rplV genes, *C. trachomatis* may also exhibit macrolide resistance [21].

Fluoroquinolones are broad-spectrum widely used bactericidal antibiotics that function by blocking the DNA gyrase and DNA topoisomerase IV enzymes needed for bacterial DNA synthesis [24]. The first-generation fluorinated quinolones include norfloxacin, ciprofloxacin, and ofloxacin. The usage of norfloxacin is restricted to the treatment of STIs and urinary tract infections because of its relatively low serum levels and inadequate tissue penetration. On the other hand, ciprofloxacin is

*Treatment of Chlamydial Infections DOI: http://dx.doi.org/10.5772/intechopen.109648*

an effective antibiotic that is still being widely used for the treatment of a number of gram-negative systemic infections. The more recent fluoroquinolones, such as levofloxacin (an isomer of ofloxacin) and moxifloxacin, have improved efficacy against gram-positive respiratory tract infections [25]. Fluoroquinolones generally have good activity against chlamydial infections; however, *in vitro* experiments have shown that sub-inhibitory antibiotic concentrations may lead to resistance in various *Chlamydia* spp. including *C. trachomatis*. It was discovered that mutations in the gyrA, parC, and ygeD genes may cause *C. trachomatis* to become resistant to fluoroquinolones, while changes in the gyrA gene may cause *C. pneumoniae* to become resistant [21].

Bactericidal drugs known as rifamycins selectively bind to the β-subunit of RNA polymerase, which result in the inhibition of the transcription process. Although they have strong *in vitro* activity, these medicines are not the first-line treatments for chlamydial infections. *In vitro* studies have demonstrated that *Chlamydia* spp., such as *C. pneumonia*, *C. trachomatis*, and *C. psittaci*, rapidly establish a resistance to rifamycins following exposure to sub-inhibitory antibiotic concentrations [3, 26]. In most research, rifampin (RIF) serves as a representative of rifamycins and rifampin resistance mostly results from rpoB gene nucleotide mutation [21].

Resistance to lincomycin, a bacteriostatic protein synthesis inhibitor, is noted in *in vitro*-generated *C. trachomatis* strains exposed to sub-inhibitory antibiotic concentrations. It was revealed that resistant strains exhibit mutations in 23S rRNA genes [3, 27].
