Clonal diversity of Haemophilus Influenzae carriage isolated from under the age of 3 years children

Objectives: Pharyngeal carriers such as H. influenzae seem to constitute the only reservoir and probably the only transmission vehicle of the invasive disease. Due to lack of information about nasopharyngeal isolation rate, epidemiology, antimicrobial resistance and genetic diversity of H. influenzae isolate from carries were assigned as main goals. Sampling was carried out as nasopharynx swabs among children less than 6 years old volunteers. After traditional biochemical tests, isolates were confirmed by targeting omp6 sequence. Following the susceptibility tests, genomic diversity of strains was analyzed by Pulsed-Field Gel Electrophoresis procedure Results: Out of 328 nasopharynx swabs, 73 strains were identified as H. influenzae. Susceptibility testing showed that levofloxacin is the most effective antibiotic against and the least effect belonged to tetracycline. Resistance to chloramphenicol (42.2%) and ampicillin (43.3%) was also observed. By genomic analysis of selected H. influenza, 28 PFGE patterns were achieved among which 11 patterns included at least 2 strains. All strains clustered into 25 different clones. The dendrogram analysis of the isolated H. influenzae strains showed that some of these strains had a clonal relationship and common genetic origin. According to our results, antibiotic resistance didn’t show any significant correlation with the clonality of strains.


Introduction
Haemophilus influenzae as an opportunistic pathogen is responsible for one third of bacterial pneumonia cases among children of 4 months to 4 years old [1]. Most healthy H.
influenzae pharyngeal carriers are children in south eastern countries who have not vaccinated against of H. influenzae. According to reports, the rate of carriage of H.
influenzae in infants are about 20% and in children 5 to 6 years old are >50% [2].
Incorrectly using antibiotics and excessively prescribing drugs are considered as one the most important factors of resisting to antibiotics. This causes therapeutic failures and continues existence of resistant strains and so further spread of the disease in medical centers [3][4][5][6][7]. H. influenzae is a bacterium that resistance to antibiotics such as cotrimoxazole, ampicillin, tetracycline, amoxicillin, cefixime, and ciprofloxacin have been growing and worrying [8][9][10]. The survival of resistant strains usually results in the fast spread of the resistant types of bacteria and finally the spread of diseases. Therefore, it is very useful to study the trend of clonal spread and trace the origin of strains in epidemiologic studies [11][12][13]. Among methods used in studying the clonal spread and genetic similarity between the strains of bacteria, we can refer to Pulsed Field Gel Electrophoresis (PFGE), which is used as a golden standard method for all bacteria such as H. influenzae [14]. In this method the whole genome of bacteria is digested and the

Reconfirmation of suspected isolates by PCR
Suspected growth colonies were screened based on the targeting a encoding external specific protein sequence termed toompP6. This sequence as conserved sequences were determined in both encapsulated and non-encapsulated H. influenzae [19]. In short. Crude DNA of suspected isolates directly was extracted taking advantage of a commercial high pure extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufactures recommendation. Quantity of extracted DNA was measured by NanoDrop ND-1000® Spectrophotometer (NanoDrop, Wilmington, DE, USA) and adjusted to 500 ng μL-1. Amplification was done on Gene Amp PCR system (Applied Biosystem, USA) by uniplex PCR method in total volume 25 μl containing; 12 μl master amplicon (Biolab, New England, UK), 1 pmol of each forward and reverse primers, a 1 μl of crude DNA as the template and 10 μl distilled water. Using program an initial cycle of denaturation94°C for

Studying Molecular Epidemiology
For studying molecular epidemiology of the collected strains, Pulsed Field Gel Electrophoresis (PFGE) was used. The protocol was described previously by Saito et al.    On the other hand, due to the pathogenic importance, H. influenzae has widely considered in epidemiologic studies on genetic similarities [19]. Given the dendrogram of the studies strains (Fig. 2), it is observed that a number of strains categorized in one clone (e.g. clone A and B). As the studied population was children under 6 years old from different centers, it is concluded that these strains circulated among children. The strains of clone A, isolated from five different children, are identical in terms of resistance to antibiotics except chloramphenicol. As the same the strains of clone B have identical resistance, except for chloramphenicol which is different in one strain. Regardless of intermediate resistance, the strains of clones D, E, F, G, and H have the same resistance pattern. The strains of clones I and J are partially different in their resistance. (Fig. 2).
The resistance pattern of one strain in clone B is identical to other strains of that clone except chloramphenicol. In terms of clone C, the strain (strain No. 89) that closely related to other strains, is different from them in resistance pattern. This may be because of the difference in the genetic algorithm. In the strains forming clone A and B, susceptibility to ceftriaxone, levofloxacin, ciprofloxacin and cefotaxime was observed. This showed that these antibiotics have a good therapeutic effect on these strains.
As the studied strains showed a less percentage of resistance to these antibiotics, these drugs can be a good choice for Haemophilus-related infections. Considering chloramphenicol and ampicillin resistant samples, the strains resistant to ampicillin are more identical in terms of genetic similarities and showed more clones comparing with strains resistant to chloramphenicol. This may mean that strains resistant to ampicillin are more resistant to common treatment than strains resistant to chloramphenicol. As seen in

Limitations
The dendrogram analysis of Haemophilus strains showed that some of these strains had clonal relations and common genetic origin. It was also observed that strains resistant and susceptible to ampicillin and chloramphenicol can be placed in the same or different clones. In fact, the patterns of antibiotic resistance did not show a significant correlation with the clonality of the strains. Chloramphenicol resistance transferring in these strains may be due to horizontal transmission. It seems that more research is needed to find the relationship between clonality and resistance of bacteria to antibiotics.

Declarations
Ethics approval and consent to participate: This study was approved by Pasteur institute of Iran ethics committee All performed on the enlarged ethical statement B 9008 meeting number in Pasteur institute of Iran. In this study, all ethics including Ethics and Consent to participate from their parents have been collected in the research.

Consent to publication: Not applicable
Availability of data and material: All the results of this study have been classified and maintained by the dissertation in the Pasteur Institute of Iran. We have indeed provided all raw data on which our study is based. Competing Interests: The authors declare that they have no competing interests.
Funding: Not applicable.