Antibiogram and Genetic Characterization of Carbapenem-Resistant Gram-Negative Pathogens Incriminated in Healthcare-Associated Infections

Abstract

Purpose: Carbapenems are considered the most efficient antibiotic used in the treatment of nosocomial infections. Carbapenem-resistant Gram-negative rods are becoming a serious hazard in hospitals threatening public health. The aim of the current study was to investigate the prevalence of carbapenem-resistant Gram-negative pathogens incriminated in healthcare-associated infections, along with antimicrobial resistance profiles, carbapenemase and metallo-β-lactamase production, and their molecular characterization.

Methods: A total of 186 clinical specimens were collected from 133 patients at various hospitals in Cairo, Egypt. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, detection of carbapenemase production using the modified Hodge test (MHT), the metallo-β-lactamase production using the EDTA combined disc test (CDT), and PCR-based detection of the bla _KPC and bla _GES resistance genes. The identification of the highly resistant retrieved isolates was then confirmed by 16S rRNA gene sequencing.

Results: The most common isolated Gram-negative species was Klebsiella pneumoniae (40.9%), followed by Acinetobacter baumannii (18.8%), Pseudomonas aeruginosa (17.3%), Escherichia coli (15.4%), Enterobacter aerogenes (5.3%), and Proteus mirabilis (2.4%). The prevalence of carbapenem-resistant isolates was 36.1% (n=75). However, 86.5% of the recovered clinical isolates were susceptible to colistin. The MHT revealed that 33.6% (n=70) of the tested strains were positive for carbapenemase production, while the CDT showed that 33.17% (n=69) of the examined strains were metallo-β-lactamase producers. The PCR revealed that 98.6% (74/75) of the tested strains possessed the bla _KPC gene; moreover, 97.3% (73/75) of the examined strains harbored the bla _GES gene.

Conclusion: This study displayed the emergence of carbapenem-resistant Gram-negative pathogens incriminated in healthcare-associated infections. The accurate identification of carbapenem-resistant bacterial pathogens is pivotal for the treatment of patients, in addition to propelling appropriate contamination control measures to restrain the fast spread of such pathogens. Colistin showed a potent in vitro antimicrobial activity against the carbapenem-resistant strains.

Keywords: 16S rRNA gene; antibiotic resistance; blaGES; blaKPC; carbapenem-resistance; gram-negative rods.

Figure 1

(A) Modified Hodge Test; Positive result appeared as a cloverleaf-like indentation of the zone of inhibition of the E. coli ATCC 25922 (indicator organism) along with the streak of inoculum. (B) Combined Disk Test; An inhibition zones with ≥ 17 mm in the inhibition zone diameter of EDTA-containing imipenem disc compared to imipenem disc alone was a positive MBL producer. The negative result appeared as no difference in the zones diameter around the two discs.

Figure 2

Gel electrophoresis analysis; (A) bla_KPC gene (638bp); M 100 bp DNA ladder; C: Negative control; lane 1: Positive control; Lanes: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13: positive isolates for bla_KPC gene with specific amplicon size 638 bp; Lane 8: negative isolate. (B) bla_GES gene (323 bp); M 100 bp DNA ladder; lane 1: Positive control; Lanes: 2–6 positive isolates for bla_GES gene with specific amplicon size 323 bp.

Figure 3

Dendrogram of the carbapenem-resistant bacterial pathogen isolated from clinical samples, constructed based on the 16S rRNA gene sequences. Bootstrap support value above branches, newly generated sequences of the isolated carbapenem-resistant Gram-negative bacterial pathogens in bold.