**4. Antibiotic resistance and heteroresistance**

#### **4.1 Antibiotic resistance**

The antibiotics once regarded as the first choice for *Hp* infection (such as metronidazole and clarithromycin), included in all therapeutic regimens, are now declining in efficacy because of their extensive use in many areas for unrelated infections. Metronidazole (MZ) mostly showed a very high resistance worldwide achieving a level up to 78.2% in China [26]. Clarithromycin (CLA) resistance rates have currently reached high levels, such as 30% in Italy and Japan, 40% in Turkey, and more than 70% in China, although rates in Sweden, Taiwan, and Germany are lower [27]. CLA was once considered the most powerful antibiotic for *Hp* infection. The local pattern of *Hp* resistance to CLA results as being crucial in each area, considering that in countries where CLA resistance is above 15–20%, this drug should not be used. CLA-resistant *Hp* has been extensively studied: its prevalence has become increasingly higher in many geographical areas. Fluoroquinolone (the most common fluoroquinolone is Levofloxacin LEV) resistance has been increasing worldwide in recent years achieving 20% in Italy, 13.3% in Germany, and 19.2% in China [26, 28]. These data are especially important in those regions planning a levofloxacin-based therapy because resistance to fluoroquinolones generally shows a major impact on the success of treatment [29].

In a study conducted in an Academic Hospital in Rome by our group on 80 *Hp* strains isolated from 80 biopsy samples in infected patients, the resistance percentage measured phenotypically through MIC values to MZ, CLA, LEV, TE (tetracycline) and AMX (amoxicillin) resulted as follows: 61.6% (50/80), 35% (28/80), 20% (16/80), 2.5% (2/80), and 1.25% (1/80), respectively. The MICs (Minimum Inhibitory Concentrations) resulted as being very high mainly to MZ showing three strains with MIC = 256 μg/mL and to CLA showing four strains with MIC ranging from 64 μg/mL to 128 μg/mL. As for LEV the MIC ranged from 0.25 μg/mL to 32 μg/mL (only one strain showed a MIC equal to 32 μg/mL) [30].

Multidrug resistance (the contemporaneous resistance to two or more antibiotics) is reported in **Figure 4**.

*Virulence Markers, Genotypic versus Phenotypic Resistance and New Treatment Strategies... DOI: http://dx.doi.org/10.5772/intechopen.97026*

#### **Figure 4.**

*Characteristics of phenotypic antibiotic resistance in* Helicobacter pylori *strains. From Mascellino et al. [30]. Notes: Proportion of phenotypic resistant* Helicobacter pylori *isolates (%) to the respective antibiotics in an Academic Hospital in Rome (Italy). Abbreviations: MZ: metronidazole; CLA: clarithromycin; LEV: levofloxacin; TE: tetracycline. Resistance belonging to the association CLA + MZ was the highest one being detected in 30% of samples (24/80), resistance to the combinations MEZ + LEV and CLA + LEV ranged between 13.75% (11/80) and 11.25% (9/80 strains) respectively. Only 6.25% samples (5/80) harbored the triple resistance MZ + CLA + LEV. As far as the dual combination MZ + TE is concerned, resistance was found just in one strain (1.25%). In China too, a similar trend of multidrug resistance of* Hp *is found [26]. In general, the antibiotic combinations consisting of MET, CLA and LEV had higher combined resistance rate, whereas the antibiotic combinations recommended in bismuth-containing quadruple therapy (Bismuth, Omeoprazole, MZ, TE) had lower combined resistance rates.*


*MZ-metronidazole, CLA-clarithromycin, AMX-amoxicillin, LEV-levofloxacin, TE-tetracycline S-susceptible, R-resistant, A-antrum, C-corpus, F-fundus. \* No growth.*

#### **Table 1.**

*Heteroresistance of 5 strains (Mascellino, unpublished data).*

#### **4.2 Heteroresistance (HR)**

Heteroresistance is defined as the concomitant presence of a different susceptibility pattern in the different districts of a single stomach in the same patient [31]. This is a common occurrence in *Hp* population and can be explained either as the result of multiple infections (unrelated isolates) or as the presence of susceptible

and resistant variants of the same strain (related isolates). In the latter case, HR has been described either as intra-district when susceptible and resistant isolates are present at the same time in the same site of gastric mucosa or as inter-district when multiple strains colonize different areas of the stomach [32, 33]. Heteroresistance of 5 strains out of 80 considered above is reported in **Table 1**.

For each of these patients, we found a different antimicrobial pattern in the strains isolated from antrum (susceptible) and corpus/fundus (resistant) towards CLA (five patients), MZ (two patients), AMX (three patients), LEV (three patients) and TE (one patient). The strain genotypes, identified on the basis of virulence genes (cagA and vacA), were the same for both loci in pairs of isolates (susceptible and resistant) obtained from different regions (A,C and F) of a single stomach (cag + s1m2 in one patient, cagA+s1m1 in three patients and cagA s2m2 in one patient).

### **5. Phenotypic resistance versus genotypic resistance**

The phenotypic resistance was calculated on the basis of MIC (Minimum Inhibitory Concentration) performed on the biopsy samples. Interpretation of susceptibility test results was performed in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2020) recommendations [34]. In order to define strains resistance the following MIC breakpoints were used: S ≤ 0.125 μg/ml and R > 0.125 μg/ml for AMX; S ≤ 0.25 μg/ml and R > 0.5 μg/ml respectively for CLA; S ≤ 1 μg/ml and R > 1 μg/ml for both TE and LEV; S ≤ 8 μg/ml and R > 8 μg/ml for MZ.

The genotypic resistance is based on the point mutations present on the chromosome. Common resistance mechanisms to clarithromycin include point mutations in the bacterial domain V of 23S rRNA, which prevents antibiotic binding [35]. There are 3 point mutations in the 23S rRNA gene: A2143G, A2142G, and A2142C; these account for 90% of cases for primary resistance in Western countries [36]. Fluoroquinolone agents (mainly levofoxacin, which is a broad-spectrum quinolone) are an alternative therapy for infections caused by *H. pylori* and serve as second-line treatment [37]. Fluoroquinolone targets are the DNA gyrase, an enzyme responsible for negative supercoiling during the DNA replication process. This enzyme contains two A subunits and two B subunits, encoded by *gyrA* and *gyrB*, respectively [38]. Resistance to clarithromycin and fuoroquinolones, which is mostly acquired through point mutations, can be detected by molecular techniques. GenoType HelicoDR (Hain Life Science, Germany) test is a molecular diagnostic method for easy and simultaneous detection of frequent point mutations responsible for clarithromycin and fuoroquinolones resistance [21].

Genotypic susceptibility testing can be also performed by a real-time PCR followed by melting curve analyses using fluorochrome labeled hybridization probes for identifying the mutations on the 16S rRNA conferring resistance to TE [28].

#### **5.1 Correlation between genotypic and phenotypic methods**

The correlation between genotypic and phenotypic methods is reported in **Table 2**.

The PCR method results as being very useful in detecting the resistant strains in comparison with the E-test technique which primarily detects the susceptible ones. Mixed *Hp* infections were demonstrated only through the PCR in patients concomitantly yielding both wild type and resistant strains in a single gastric region. The resistance with both methods (phenotypic tests and PCR molecular tests) for CLA

*Virulence Markers, Genotypic versus Phenotypic Resistance and New Treatment Strategies... DOI: http://dx.doi.org/10.5772/intechopen.97026*


*S = susceptible, R = Resistant, A2143G = point mutation on CLA23SrRNA gene conferring resistance to CLA, gyrA = gene conferring resistance to LEV.*

*\* Refers to mixed iinfections (from Mascellino et al. [39]).*

#### **Table 2.**

*Correlation between genotypic and phenotypic methods.*

and TE demonstrated the superiority of PCR over the phenotypic test (data not shown). As for CLA, the resistance was detected in 76% (16/21) of patients by PCR and 57% (12/21) by E-test, whereas for LEV the same values corresponded to 83% (10/12) and 41% (6/12) respectively. In this last case the difference turned out to be statistically significant (p < 0.05).

The genotypic test performed directly on the biopsy samples shows some great advantages over the E-test. In fact the genotypic-resistance resulted very useful to identify mixed infections that represent a real problem possibly leading to a resistance underestimation or in the case of absence of live bacteria (coccoid forms) or contamination. The real-time PCR detects the resistant population at a lower concentration than the phenotypic tests which primarily show susceptible bacteria. Following our results [30], the E-test is unable to detect all resistant strains because when there are many susceptible bacteria compared with the resistant ones, these susceptible bacteria are identified first leading to a misclassification. On the contrary through a direct examination of gastric samples by genetic tests, a diversity of strains (susceptible, resistant or both) can be detected at the same time. Mixed *Hp* infections were demonstrated in patients yielding both wild type and resistant strains in a single gastric region at the same time. The use of genotypic tests directly on the clinical specimens could predict the antibiotic resistance addressing changes in previous treatments or for evaluating the primary resistance to antibiotics (ie CLA) in order to avoid administration of ineffective antimicrobials [23, 38].
