According to the reports by world health organization (WHO), AMR is common in many countries; however, an exact estimation of the extent of the problem and the economic losses due to AMR is not available. WHO reports that Iran is categorized in the countries with more than five MDR bacteria and has a high prevalence of AMR in the selected bacteria ; however, there is no exact information on AMR in the commensal isolates in the country. In this study, to evaluate the frequency of AMR in the community, infants and children under three years old were investigated. Fetuses have sterile intestines, and they receive microorganisms from their mothers at birth. After a few minutes, the gastric content of the neonate is influenced by the received flora from the mother. Gradually, infants receive intestine flora from family members, environment, water, and food and normal intestine flora forms before three years old . Therefore, the commensal flora of this group often has not been directly exposed to antibiotics. That is why this age group was chosen and children received antibiotics were excluded from the study.
For the first time in 1966, AMR in commensal E. coli from healthy community members was reported  and further studies highlighted the increasing incidence of AMR in commensal E. coli from many countries [14, 32, 33]. E. coli is known as the main reservoir of resistance in fecal flora. In this study, the frequency of AMR in commensal E. coli isolates from two region of Iran was investigated and found that the incidence of AMR, MDR, and ESBL-producing isolates in Khuzestan Province was significantly higher as compared to Fars Province. Interestingly, according to the report by the National Committee for Rational Prescribing and use of Drugs (NCRUD) in Iran, in 2015 the highest and lowest mean numbers of prescribed antimicrobial agents were related to Khuzestan and Fars Provinces, respectively . This level of prescribed antibiotics may be due to the high prevalence of infectious diseases in Khuzestan Province. In other studies performed in Khuzestan, high frequencies of AMR, MDR, and ESBL in diarrheagenic E. coli (DEC) isolates  and ESBL-producing E. coli strains in urinary tract infections were reported . However, the frequencies of AMR, MDR, and ESBL in our study were lower than those studies. Probably, communication between pathogenic and commensal bacteria and indirect exposure of commensal flora to antibiotics caused high AMR in the community. As reported by the European Antimicrobial Resistance Surveillance Network (EARS-Net), MDR among infectious E. coli isolates ranged from approximately 1% in 2002 to 4.8% in 2016 and 10.1% in 2018 ; however, in our study, MDR was found to be 36%, which is disconcerting. The carriage of commensal AMR, MDR and ESBL-producing isolates in healthy children under three years of age mostly reflects exposure to contamination in the family environment, water, and food rather than increased direct exposure to antimicrobial drugs .
Carbapenems are highly effective broad-spectrum beta-lactam antibiotics commonly used as the last-line antibiotics for the treatment of severe or high-risk antibiotic resistant Gram-negative bacterial infections. The emergence and dissemination of carbapenem-resistance mechanisms represent a global public health concern because no solutions have been found for this problem yet . According to a report by Castanheira et al. the rates of CR in pathogenic Enterobacteriaceae increased from 0.6% in 1997–2000 to 2.9% in 2013–2016. In our study, the rate of CR in commensal E. coli strains was 1.6%; however, we do not have the exact rate of CR in pathogenic Enterobacteriacea .
The emergence of MBL-producing isolates is alarming since they carry mobile genetic elements with great ability to spread; therefore, early detection of these isolates, particularly their reservoir, is crucial to prevent their inter- and intra-care setting dissemination and establish suitable antimicrobial therapies . To find MBL-producing isolates, we used different tests including CDT, DDST, MHT, mCIM, and eCIM because no perfect test has been introduced to identify all types of MBLs. Using mCIM in combination with eCIM, we could detect four MBL-producing isolates. According to the CLSI-2018, MHT is no longer considered a reliable phenotypic method for carbapenemase detection and other methods such as the CarbaNP and mCIM have taken its place because MHT cannot detect some carbapenemase-producing isolates including NDM-producing strains . In the present study, blaNDM-positive isolates were not detected by MHT. Castanheira et al. collected pathogenic Enterobacteriaceae isolates from 42 countries over 20 years and found that the rates of MBL-carrying isolates increased from 4.3% of the CR isolates in 2007 to 2009 to 12.7% from 2014 to 2016 , while in the present study 66% of commensal CR isolates were detected as MBL-producing. PCR results showed that two and one isolates had the ability to produce NDM and VIM-2, respectively; however, one MBL-producing isolate was negative for all the investigated genes. NDM has worldwide distribution and is the most common MBL in the Enterobacteriaceae family . Based on the report by Castanheira et al., the dissemination of isolates carrying blaNDM caused increased CR among Enterobacteriaceae isolates from 2014 to 2016 . For the first time in Iran, NDM was detected in K. pneumonia in 2013 . Eyvazi et al. reported NDM-producing E. coli isolates in 2017  and NDM-producing Pseudomonas aeruginosa was isolated from filters of household water treatment systems in Ahvaz, Khuzestan, Iran . Probably in Khuzestan, some of the resistance mechanisms are transported through the water from resistant environmental bacteria to the intestinal commensal flora. The spread of isolates carrying blaVIM in Italy and Greece led to increased CR among the European countries in 2005 . There are some reports in Iran on the detection of blaVIM−2 in clinical isolates such as hypervirulent Klebsiella pneumonia or pathogenic P. aeruginosa [43, 44]. However, we did not find any reports regarding the detection of MBL genes among commensal E. coli isolates. The present results showed that the commensal E. coli isolates can harbor MBL resistance plasmids and transfer them through conjugation. All of the transconjugative colonies were resistant to imipenem; however, not all of the plasmids were transferred. The blaNDM genes are often carried by plasmids; therefore, they can easily move to other bacteria through HGT, which increases the probability of the emergence of antimicrobial resistant strains of pathogenic bacteria  as seen in the present study. The bacteria that synthesize NDM-1 are highly resistant to all antibiotics, including carbapenems and aminoglycosides ; while, two NDM-producing isolates in our research were sensitive to gentamicin an aminoglycoside. NDM-1 has several variants and the one identified in our research was related to an NDM-5 variant. This variant has a greater hydrolytic activity than NDM-1 toward carbapenems, and cephalosporins including cefotaxime, cephalotin and ceftazidime .
Out of six CR isolates, one was found phenotypically positive by PBA and mCIM tests and considered class A KPC carbapenemase. However, we could not identify the carbapenemase gene of this isolate. One CR isolate was positive only by mCIM. The resistance of this isolate is probably as class A or D of ambler classification, although this isolate was negative by the general primers of blaKPC, oxa48, and oxa23. Resistance to carbapenems can also be due to non-carbapenemase-mediated mechanisms, such as hyper production of a beta-lactamase, typical AmpC beta-lactamase, combination of ESBLs or AmpCs with porin mutations, and reduced membrane permeability [10, 20, 39].