The prevalence of Helicobacter pylori primary resistance towards antibiotics using an Epsilometer-test method

Abstract@#An epsilometer-test method was used to determine MIC values of several antibiotics against 29 Helicobacter pylori isolated from gastric antrum of dyspepsia patients. Isolates with resistance towards antibiotics were 6.9% -65.5% but these were tetracycline-sensitive. Eight isolates showed multi-resistance towards two antimicrobial agents. The high resistance strains towards metronidazole is alarming.

Distribution of gastric adenocarcinoma subtypes in different ethnicities in Kuala Lumpur, Malaysia

The multiracial population in Malaysia has lived together for almost a century, however, the risk ofgastric cancer among them varies. This study aimed to determine the distribution of different gastricadenocarcinoma subtypes and Helicobacter pylori infection status among gastric adenocarcinomapatients. Patients with gastric adenocarcinoma were enrolled from November 2013 to June 2015.Blood samples were collected for detection of H. pylori using ELISA method. Gastric adenocarcinomacases were more prevalent in the Chinese (52.8%), followed by the Malays (41.7%) and leastprevalent in the Indians (5.6%). Gastric adenocarcinoma located in the cardia was significantly moreprevalent in the Malays (66.7%) compared to the Chinese (26.3%), whereas non-cardia cancer wasdiagnosed more in the Chinese (73.7%) compared to the Malays (33.3%) [P = 0.019; OR = 5.6, 95CI: 1.27 to 24.64]. The Malays also had significantly higher prevalence of gastric tumour locatedat the cardia or fundus than other gastric sites compared to the Chinese (P = 0.002; OR: 11.2, 95%CI: 2.2 to 56.9). Among the cardia gastric cancer patients, 55.6% of the Malays showed intestinalhistological subtype, whereas all the Chinese had the diffuse subtype. More than half of the patients(55.3%) with gastric adenocarcinoma were positive for H. pylori infection and among them, 66.7%were Chinese patients. The risk of gastric adenocarcinoma in our population is different amongethnicities. Further studies on host factors are needed as it might play an important role in gastriccancer susceptibility in our population.

Molecular detection of the New Delhi metallo-ß-lactamase-1 gene in Enterobacteriaceae isolates in a tertiary medical centre.

BACKGROUND: New Delhi metallo-ß-lactamase-1 (NDM-1) is a relatively recent carbapenemase enzyme that inactivates all ß-lactam antibiotics with the exception of aztreonam. This study aims to ascertain the baseline prevalence and antibiotic susceptibility patterns of NDM-1-producing Enterobacteriaceae in a tertiary medical center in Malaysia. METHODS: Over a period of one year, all Enterobacteriaceae isolates from all clinical specimens with reduced susceptibility to at least one carbapenem and resistance to at least one third generation cephalosporin were subjected to antibiotic susceptibility testing by disk diffusion and molecular detection of the NDM-1 gene by single-target PCR followed by gel electrophoresis. RESULTS: A total of 13,098 Enterobacteriaceae isolates were screened and 63 (0.48%) had reduced susceptibility to at least one carbapenem. Of this 63, 18 (29%) were NDM-1-positive. Of this 18, 16 (89.0%) were Klebsiella pneumoniae, one (5.5%) was Escherichia coli and one (5.5%) was Klebsiella ornithinolytica. Reduced susceptibility to at least one aminoglycoside was seen in 17 (94%) of the NDM-1-positive isolates. All 18 (100%) had reduced susceptibility to ertapenem and were resistant to all the second and third generation cephalosporin antibiotics tested. CONCLUSION: The prevalence of NDM-1-producing Enterobacteriaceae among all the Enterobacteriaceae isolates in our institution is low (0.14%) and screening for the NDM-1 gene is best performed using ertapenem-impregnated disks.

Molecular characterization of carbapenemase and cephalosporinase genes among clinical isolates of Acinetobacter baumannii in a tertiary medical centre in Malaysia.

Antimicrobial resistance in Acinetobacter baumannii is a growing public health concern and an important pathogen in nosocomial infections. We investigated the genes involved in resistance to carbapenems and cephalosporins in clinical A. baumannii isolates from a tertiary medical centre in Malaysia. A. baumannii was isolated from 167 clinical specimens and identified by sequencing of the 16S rRNA and rpoB genes. The MIC for imipenem, meropenem, ceftazidime and cefepime were determined by the E-test method. The presence of carbapenemase and cephalosporinase genes was investigated by PCR. The isolates were predominantly nonsusceptible to carbapenems and cephalosporins (>70 %) with high MIC values. ISAba1 was detected in all carbapenem-nonsusceptible A. baumannii harbouring the blaOXA-23-like gene. The presence of blaOXA-51-like and ISAba1 upstream of blaOXA-51 was not associated with nonsusceptibility to carbapenems. A. baumannii isolates harbouring ISAba1-blaADC (85.8 %) were significantly associated with nonsusceptibility to cephalosporins (P<0.0001). However, ISAba1-blaADC was not detected in a minority (<10 %) of the isolates which were nonsusceptible to cephalosporins. The acquired OXA-23 enzymes were responsible for nonsusceptibility to carbapenems in our clinical A. baumannii isolates and warrant continuous surveillance to prevent further dissemination of this antibiotic resistance gene. The presence of ISAba1 upstream of the blaADC was a determinant for cephalosporin resistance. However, the absence of this ISAba1-blaADC in some of the isolates may suggest other resistance mechanisms and need further investigation.

Screening for New Delhi metallo-ß-lactamase-1 in Enterobacteriaceae: Is there a role for the modified Hodge test?

OBJECTIVE: The New Delhi metallo-ß-lactamase-1 (NDM-1) enzyme is a plasmid-encoded enzyme that inactivates carbapenem antibiotics. This study aims to ascertain if the modified Hodge test (MHT) has a role in screening for NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility. METHODS: Over a period of one year, all Enterobacteriaceae isolates from all clinical specimens with reduced susceptibility to at least one carbapenem were subjected to MHT and conventional polymerase chain reaction (PCR) detection of the NDM-1 gene. RESULTS: A total of 13,098 Enterobacteriaceae isolates were screened and 63 (0.48%) had reduced susceptibility to at least one carbapenem. Out of the 63 isolates, 45 (71.4%) were MHT-positive. The NDM-1 gene was detected in 18 of the 63 isolates (28.6%). All 18 PCR-positive isolates were also MHT-positive. Thus, the sensitivity and specificity of the MHT in detecting NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility are 100% and 40%, respectively. CONCLUSION: The MHT is a useful test to screen for the presence of NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility. However, due to its rather low specificity, all MHT-positive isolates should be subjected to alternative tests (e.g. PCR) for confirmation, especially if other types of carbapenemases (e.g. KPC) are prevalent.

Analysis of clinical isolates of Helicobacter pylori in Pakistan reveals high degrees of pathogenicity and high frequencies of antibiotic resistance.

BACKGROUND: Antibiotic resistance in Helicobacter pylori contributes to failure in eradicating the infection and is most often due to point and missense mutations in a few key genes. METHODS: The antibiotic susceptibility profiles of H. pylori isolates from 46 Pakistani patients were determined by Etest. Resistance and pathogenicity genes were amplified, and sequences were analyzed to determine the presence of mutations. RESULTS: A high percentage of isolates (73.9%) were resistant to metronidazole (MTZ), with considerable resistance to clarithromycin (CLR; 47.8%) and amoxicillin (AML; 54.3%) also observed. Relatively few isolates were resistant to tetracycline (TET; 4.3%) or to ciprofloxacin (CIP; 13%). However, most isolates (n = 43) exhibited resistance to one or more antibiotics. MTZ-resistant isolates contained missense mutations in oxygen-independent NADPH nitroreductase (RdxA; 8 mutations found) and NADH flavin oxidoreductase (FrxA; 4 mutations found). In the 23S rRNA gene, responsible for CLR resistance, a new point mutation (A2181G) and 4 previously reported mutations were identified. Pathogenicity genes cagA, dupA, and vacA s1a/m1 were detected frequently in isolates which were also found to be resistant to MTZ, CLR, and AML. A high percentage of CagA and VacA seropositivity was also observed in these patients. Phylogenetic analysis of partial sequences showed uniform distribution of the 3' region of cagA throughout the tree. CONCLUSIONS: We have identified H. pylori isolates in Pakistan which harbor pathogenicity genes and worrying antibiotic resistance profiles as a result of having acquired multiple point and missense mutations. H. pylori eradication regimens should therefore be reevaluated in this setting.

Resistotype of Helicobacter pylori isolates: the impact on eradication outcome.

Antibiotic resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. The aim of this study was to determine the phenotype and genotype of antibiotic-resistant strains of H. pylori in the Malaysian population and to evaluate the impact of antibiotic resistance to eradication outcome. One hundred and sixty-one H. pylori isolates were analysed in this study. Metronidazole, clarithromycin, fluoroquinolone, amoxicillin and tetracycline susceptibilities were determined by Etest. PCR followed by DNA sequencing was carried out to determine mutations. The medical records of the patients infected with resistant strains were reviewed to determine the eradication outcome. Metronidazole resistance was encountered in 36.6 % of H. pylori isolates, whereas clarithromycin and fluoroquinolone resistance was observed in 1.2  and 1.9 % of isolates, respectively. All strains tested were susceptible to amoxicillin and tetracycline. Frameshift and nonsense mutations in rdxA and frxA genes resulting in stop codons contributed to metronidazole resistance, which leads to reduced eradication efficacy. A2142G and A2143G mutations of 23S rRNA were identified as causing failure of the eradication therapy. Mutation at either codon 87 or 91 of the gyrA gene was identified in fluoroquinolone-resistant strains. However, the effect of resistance could not be assessed. This study showed that frameshift and nonsense mutations in rdxA or frxA genes and point mutations in the 23S rRNA affected the efficacy of H. pylori eradication therapy.

Ethnicity association of Helicobacter pylori virulence genotype and metronidazole susceptibility.

AIM: To characterise the cag pathogenicity island in Helicobacter pylori (H. pylori) isolates by analysing the strains' vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome. METHODS: Ninety-five H. pylori clinical isolates obtained from patients with dyspepsia living in Malaysia were analysed in this study. Six genes in the cagPAI region (cagE, cagM, cagT, cag13, cag10 and cag67) and vacA alleles of the H. pylori isolates were identified by polymerase chain reaction. The isolates' metronidazole susceptibility was also determined using the E-test method, and the resistant gene was characterised by sequencing. RESULTS: More than 90% of the tested isolates had at least one gene in the cagPAI region, and cag67 was predominantly detected in the strains isolated from the Chinese patients, compared with the Malay and Indian patients (P < 0.0001). The majority of the isolates (88%) exhibited partial deletion (rearrangement) in the cagPAI region, with nineteen different patterns observed. Strains with intact or deleted cagPAI regions were detected in 3.2% and 8.4% of isolates, respectively. The prevalence of vacA s1m1 was significantly higher in the Malay and Indian isolates, whereas the isolates from the Chinese patients were predominantly genotyped as vacA s1m2 (P = 0.018). Additionally, the isolates from the Chinese patients were more sensitive to metronidazole than the isolates from the Malay and Indian patients (P = 0.047). Although we attempted to relate the cagPAI genotypes, vacA alleles and metronidazole susceptibilities to disease outcome, no association was observed. The vacA alleles were distributed evenly among the strains with intact, partially deleted or deleted cagPAI regions. Interestingly, the strains exhibiting an intact cagPAI region were sensitive to metronidazole, whereas the strains with a deleted cagPAI were more resistant. CONCLUSION: Successful colonisation by different H. pylori genotypes is dependent on the host's genetic makeup and may play an important role in the clinical outcome.

Association of Malaysian Helicobacter pylori virulence polymorphisms with severity of gastritis and patients' ethnicity.

BACKGROUND AND AIM: Polymorphisms of Helicobacter pylori cagA and vacA genes do exist and may contribute to differences in H. pylori infection and gastroduodenal diseases among races in the Malaysian population. This study was conducted to characterize the polymorphisms in H. pylori cagA and vacA in Malaysian population. METHODS: A total of 110 H. pylori isolates were genotyped by PCR and sequenced for cagA and PCR-RFLP for vacA. RESULTS: East Asian cagA was predominantly detected (64.5%), whereas vacA s1m1 and s1m2 alleles were detected in 60.9 and 37.3% of strains, respectively. A statistical association between cagA type with patients' ethnicity (p < .0001) and age group >50 years old (p = .027) was identified. vacA alleles showed significant association with age group >50 years old (p = .017) and increased neutrophil activity in gastric mucosa (p = .028 and p = .016 for moderate and marked activity, respectively). Further identification of vacA polymorphism revealed that 84% of strains from Malays and Indians showed one RFLP pattern (RFLP-1), whereas more than one RFLP patterns (RFLP-2, 3, 4, 5, 6, and 8) were predominantly observed in strains from Chinese (82%) (p < .0001). Increasing severity of gastric inflammation was observed in gastric mucosa infected with strains carrying RFLP-2, 3, 4, 5, and 6 (p = .037). About 86.6% of H. pylori strains with East Asian cagA were vacA RFLP-2, 3, 4, 5, 6, and 8, and 88% of Western cagA strains were vacA RFLP-1 (p < .0001). Chinese and Indians are susceptible to different virulence genotypes of H. pylori, whereas Malays showed a mixed virulence genotypes. CONCLUSION: Marked differences in the polymorphisms of cagA and vacA were observed among strains in Malaysian population. This provides a new insight into the pathogenicity of H. pylori in multiracial population.

Variant of Helicobacter pylori CagA proteins induce different magnitude of morphological changes in gastric epithelial cells.

Infection with Helicobacter pylori cagA-positive strains is associated with gastroduodenal diseases. The CagA protein is injected into gastric epithelial cells and supposedly induces morphological changes termed the 'hummingbird phenotype', which is associated with scattering and increased cell motility. The molecular mechanisms leading to the CagA-dependent morphological changes are only partially known. The present study was carried out to investigate the effect of CagA variants on the magnitude of gastric epithelial cell morphological changes. Recombinant 3' terminal domains of cagA were cloned and expressed in a gastric epithelial cell line and the hummingbird phenotype was quantified by microscopy. The 3' region of the cagA gene of Malaysian H. pylori isolates showed six sub-genotypes that differed in the structural organization of the EPIYA repeat sequences. The percentage of hummingbird cells induced by CagA increased with duration of transfection. The hummingbird phenotype was observed to be more pronounced when CagA with 4 EPIYA motifs rather than 3 or 2 EPIYA motifs was produced. The activity of different CagA variants in the induction of the hummingbird phenotype in gastric epithelial cells depends at least in part on EPIYA motif variability. The difference in CagA genotypes might influence the potential of individual CagAs to cause morphological changes in host cells. Depending on the relative exposure of cells to CagA genotypes, this may contribute to the various disease outcomes caused by H. pylori infection in different individuals.

cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groups.

Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3' region of cagA gene were examined among 192 Malaysian H. pylori cagA-positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621-651bp), type B (732-735bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p>0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific-cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p<0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p<0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection.