Pathogenesis and Drug Resistance in Haemophilus parainfluenzae

Bacteria Haemophilus parainfluenzae

Haemophilus parainfluenzae (HPi) is a nonmotile, nonspore-forming, microaerophilic, Gram-negative, small rod-shaped bacterium. It is usually coccobacilli or filamentous in form. The optimum temperature for growth is 35-37°C, which is typical of its habitat in the respiratory tract. In vitro, HPi requires V factor for growth under aerobic conditions but does not need X factor. It is non-hemolytic, can ferment glucose and sucrose producing acid without gas, does not ferment lactose, and tests positive for catalase. These characteristics help distinguish HPi from other bacteria.

HPi can be subtyped into 8 biotypes (I to VIII) and one untypeable biotype based on differences in 3 biochemical reactions (ornithine decarboxylase, urease and indole production). The prevalence of biotypes is quite different in healthy carriers and patients with lower respiratory tract infections. In healthy people, the throat harbors a diverse mix of HPi biotypes, often with several types found in the same sample. However, in disease states, strains isolated from lower respiratory tract secretions are usually of a single biotype. It has been found that biotypes I and II appear to be the cause of most respiratory infections. Biotypes isolated from URTI, biotype I is the most common, followed by biotype II. The ratio of biotype I decreases and that of biotype II increases as the site of infection is lower.

HPi is a frequent human opportunistic pathogen of the respiratory tract, from which a variety of diseases (endocarditis, nephritis, genitourinary infections, biliary tract infections, peritonitis) may occur. HPi is considered a commensal primarily in the upper respiratory tract, predominantly in the oropharynx. It has been demonstrated that in the presence of local or systemic immunity deficiency or bacterial imbalance, HPi may penetrate and infect the lower respiratory tract. Studies dating back to the last century suggested that HPi can produce histamine and other inflammatory mediators, making it a potential pathogen in lower respiratory tract infections like acute exacerbations of chronic bronchitis, cystic fibrosis, and pneumonia. Surveys comparing HPi carriage in people with lower respiratory infections and healthy individuals have found a high prevalence of HPi in both groups.

Antibiotic Resistance

In light of the increasing usage of broad-spectrum antibiotics, resistance to HPi has also shown to be gradually increasing. Plasmid mediated production of β-lactamase is the most common cause of HPi resistance. HPi is capable of producing two types of β-lactamase, TEM-1 and ROB-1. Non-enzymatic mechanisms of resistance include changes in penicillin-binding proteins (PBPs) that reduce ampicillin binding to PBPs and alterations of bacterial outer membrane protein permeability that enable the bacteria to continue to multiply in the presence of antimicrobial agents.

Table 1. Average resistance in H. influenzae and H. parainfluenzae isolates among patients with CF between 1998 and 2012

(Source: Ebbing, Rosanne, et al. 2015)

• β-lactam antibiotics

Resistance to β-lactam antibiotics is commonly mediated by four mechanisms: by production of β-lactamases that synthesize and export β-lactam antibiotic hydrolyzing enzymes; by alteration of the outer membrane permeability, often by porin modification; by synthesis of altered PBPs with a lower affinity for the antibiotic; and by the expression of efflux pumps that actively expel antibiotics from the cell. Since the first report of β-lactam resistance in HPi by Groves et al in 1976, isolates with β-lactam resistance have been reported from all over the world. The β-lactamases they express have been shown on frequent occasion to be encoded by transferable plasmids, conferring not only β-lactam resistance to the organism itself but making H. parainfluenzae a potential important source of transfer of β-lactamase genes to other species. The most common types of β-lactamases expressed by H. parainfluenzae are TEM-1 and TEM-15, which are both plasmids associated. The inhibitor-resistant β-lactamases TEM-34 and TEM-182, both carried on Tn2-type transposons, were discovered in HPi in 2011.

In addition to β-lactamase-dependent mechanisms, HPi strains also have the ability to display non-enzymatic mechanisms, the majority of which result from mutations in PBPs. PBP mutation results in a structural change of PBP, which leads to a lower affinity for the antibiotic, and thus prevents the antibiotic from working. Amino acid substitutions have been identified in the PBP3 protein of HPi through molecular biology methods. Lys276Asn, Ala307Asn, and Val329Ile were some of the substitutions that were identified as playing a key role in bacterial tolerance to β-lactam agents. Although efflux pumps and porin expression are two common mechanisms of drug resistance in many organisms, no evidence supports the involvement of the two mechanisms in β-lactam resistant strains of HPi.

• Macrolides, azalides, ketolides, lincosamides and streptogramins

HPi has been shown to acquire resistance to all of these classes of antibiotics through three basic mechanisms. One is an active efflux system in which efflux pumps encoded by genes such as msr(A), msr(B), msr(D), and mef(A) actively remove antibiotics from the bacterial cell. A second way is through drug inactivation in which enzymes, such as esterases and phosphotransferases, encoded by mph genes, chemically modify the antibiotic and destroy it. The third and most important mechanism is targeting modification, primarily achieved through methylation. This methylation is usually mediated by methyltransferases encoded by erm genes, which alter the antibiotic binding sites. Additionally, mutations in the V region of 23S rRNA and ribosomal proteins L4 and L22 can block antibiotic binding to their ribosomal targets.

HPi has several ways to resist tetracycline antibiotics such as: enzymatic inactivation of tetracycline, rRNA mutations, efflux pump-mediated resistance, ribosomal protection proteins production and others poorly understood. The most significant mechanism is the production of ribosomal protection proteins such as Tet(T), Tet(S), Tet(Q), Tet(B), Tet(W), Tet(O), Tet(M), and OtrA.

Molecular detection of fluoroquinolone-resistant HPi reveals that resistance is most frequently due to mutations in the genes that encode the target proteins, DNA gyrase and topoisomerase IV. Mutations in these genes result in amino acid substitutions in QRDRs.

The primary methods of Aminoglycoside resistance are: increased activity of efflux pumps, reduced outer membrane permeability, mutation of target molecule, and enzymatic inactivation (Enzymatic inactivation is the most common). The involved enzymes are usually phosphotransferases, acetyltransferases, and nucleotidyltransferases. The resistance of HPi to the aminoglycosides has been reported. The underlying molecular mechanisms are not yet clear and need further study.

Resistance of H. parainfluenzae to antifolate drugs such as sulfonamides and trimethoprim involves several mechanisms, including the use of alternative metabolic pathways, increased cell wall impermeability, production of resistant chromosomal enzymes, overexpression of sensitive chromosomal enzymes, and plasmid-mediated resistant enzymes. Plasmid- and transposon-carried sulfonamide (sul) and trimethoprim (dfr) resistance genes have been reported. Additionally, mutations in the chromosomal folP gene, which encodes dihydropteroate synthase—such as Leu186Phe, Asp238Asn, Asn245Lys, and Phe246Tyr—reduce the enzyme's affinity for sulfonamides, thereby enhancing resistance.

Table 2. Molecular resistance mechanisms of H. parainfluenzae

(Source: Abotsi, R. E., et al. 2017)

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