Intestinal flora forms a major reservoir of bacteria, including multidrug-resistant bacteria. In this study, the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45%, and the rate of carrying CRE was 1.6%, of which the dominant species were K. pneumoniae and E. coli, consistent with a previous report [10]. This showed that K. pneumoniae and E. coli were the dominant species in 60 CRE isolated from 704 stool samples. However, the rate of feces carrying CRE was 8.5%; this may be due to the fact that the stool samples collected in this study were primarily from hospitalized patients in Intensive Care Unit. The fecal samples collected in the present study contained 5.0% carbapenem-resistant non-fermenting bacillus, and Acinetobacter spp. was the dominant species accounting for 2.3%; this result was similar to our previous report of the positive rate of Acinetobacter spp. [11]. In general, the abundance of Enterobacteriaceae in the normal intestine is greater than Acinetobacter, but the positive rate of carbapenem-resistant Acinetobacter in the intestine was higher than Enterobacteriaceae in our study. Acinetobacter is the main pathogenic bacteria that causes nosocomial infections and is widely distributed in the environment, which is significant for the spread of drug-resistant bacteria. We isolated 22 genera of CR-GNB from stool samples, some of which did not cause clinical infections, suggesting the possibility that drug resistance had spread widely between different strains.
CR-GNB isolates in the present study exhibited high resistance rates to β-lactam antimicrobial agents, including cephalosporins, carbapenems, and the monobactam aztreonam; however, most isolates were sensitive to colistin. The susceptibility of different CRE species to amikacin varied; most of the isolates exhibited low resistance to amikacin (< 20.0%). Liu et al. [10] reported that resistance to amikacin against CRE species isolated from fecal samples was less than 25%, while Wang et al. [12] reported that 1801 clinical CRE isolates showed susceptibility to amikacin (54.5%). Carbapenem-resistant strains isolated from fecal survey samples were more sensitive to amikacin than clinical strains. Pritsch et al. [13] reported that two of three carbapenamase-producing A. baumannii isolates were untreatable with locally available antimicrobial agents and were susceptible to colistin and amikacin only. In some cases, Our datas provided an effective choice for clinical treatment of infections.
A total of 307 (45.1%) isolates carried carbapenemase genes. blaNDM (67.1%, 206/307) was the predominant carbapenemase gene in the present study; the four main types included blaNDM−1, blaNDM−4, blaNDM−5, and blaNDM−7. The positive rate of the blaNDM−1 gene was higher than for any other variant, consistent with previous reports [14]. In the present study, blaNDM was identified in different species, and the sequencing of four different species indicated that blaNDM can be transmitted between different species through mobile genetic elements or removable plasmids, including Enterobacteriaceae and non-fermenting Bacillus. Notably, NDM was mainly present in Acinetobacter, and we detected only one type of carbapenemase NDM in Acinetobacter, indicating that NDM production is an important factor in the resistance of Acinetobacter to carbapenem. Other metal enzymes such as VIM and IMP were mainly present in Pseudomonas spp., and KPC was mainly present in K. pneumoniae. Apart from the NDM, KPC, VIM, and IMP carbapenemases, no other carbapenemase genes were found. OXA-48, a carbapenemase gene found in many European countries, is rare in China. Whether the results of the present study, and those of previous studies in which OXA-48 was not detected, are typical, will require further investigation [15, 16]. In addition, 373 carbapenemase gene-negative GNB isolates were identified. Other mechanisms of carbapenem resistance have been recognized, which include overexpression of Extended Spectrum Beta-Lactamases (ESBLs) or AmpC β-lactamase, decreased membrane permeability due to porin loss, and expression of efflux pumps [17, 18]. The complexity of carbapenem resistance mechanisms in GNB and characteristics of the human gastrointestinal tract make the dissemination and transmutation of resistance genes more complex and frequent.
The blaAFM gene was discovered in our research. This gene is a novel metallo-β-lactamase gene; its expression product can hydrolyze carbapenemase. Therefore, strains carrying the blaAFM gene can also develop resistance to carbapenem antibiotics. In the present study, the blaAFM gene was also present in different strains of the human gastrointestinal tract. Although it was not detected in common isolates of Enterobacteriaceae, the blaAFM gene is often located on a plasmid and can spread through mobile genetic elements.
Colistin is a last-resort antimicrobial for infections caused by multidrug resistant Gram-negative bacteria. In 680 CNS-GNB, six isolates were resistant to colistin, which indicated low prevalence of the mcr-1 gene among carbapenemase-producing fecal survey isolates. The mcr-1 gene is a transferable resistance determinant against colistin. We detected five strains of Acinetobacter and one strains of K. pneumoniae co-producing mcr-1 and NDM-5 in the present study. It has been reported that both blaNDM and mcr genes are commonly present in E. coli isolated from different sources, but the co-existence of the blaNDM and mcr genes in K. pneumoniae has rarely been reported. In this study, the plasmid harboring the mcr gene in K. pneumoniae was similar to other IncHI2-type plasmids present in E. coli, and upstream of the mcr gene was the common ISApl1 transposon. NDM-5 has been reported in many other countries, including India [19], China [20], Denmark [21], Italy [22], America [23], and Spain [24]; it has also been isolated from pigs [25, 26], dairy cows [27], and vegetables [28]. In the present study, the IncX3 plasmids harboring blaNDM−5 were highly similar in various sources, suggesting their ability to be an efficient vehicle for blaNDM−5 dissemination among humans, animals, food, and the environment via the human intestine. This strain can capture the plasmid harboring mcr or blaNDM genes, which leads to the generation of pan-drug resistance, and the phenomenon of co-existence of mcr or blaNDM genes has spread to other isolates, which poses a significant challenge in the treatment of clinical infections.
In the present study, individuals with CRE were primarily from the pneumology department, hematology department, intensive care unit, and rehabilitation department. There were some common feature of individuals with CRE, most of them had infectious diseases, multiple hospital admissions and higher infectious risk, the history of using antibiotics. Therefore, the frequent use of antibiotics was closely related to multidrug resistant bacteria. However, some individuals with CRE did not have underlying diseases, and there was no record of previous antibiotic use. In addition, these individuals were from different regions of China, indicating that CRE has been widely present in the intestines of normal individuals. If individuals carrying resistance genes have infections, failure of antibiotic treatment, as well as an increase in the risk of transmission to other patients, is likely.
In conclusion, the emergence of CNS-GNB in fecal matter poses a major concern. Our data showed that the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45%, Acinetobacter was the dominant isolate of CNS-GNB, and most of the strains were sensitive to colistin. The presence of carbapenemase genes was the main mechanism of CNS-GNB resistance to carbapenem antibiotics, mainly encoding NDM. Notably, blaNDM genes were widely distributed in various isolates. The blaNDM genes were first detected in Providencia vermicola, Achromobacter spp., and Cupriavidus gilardii, and Achromobacter co-producing blaVIM and blaIMP genes has not been reported. Genomic sequencing and analysis of K. pneumoniae harboring blaNDM and mcr revealed that these genes were located on two different plasmids, which were similar to previously reported plasmids, suggesting their ability to be efficient vehicles for blaNDM−5 dissemination among humans, animals, food, and the environment via the human intestine. Our results highlight the fact that enhanced surveillance and health policies for the detection and control of these pathogens are urgently needed to limit the emergence and spread of such an organism. As the final antibiotic for the treatment of CR-GNB infection, the emergence of MCR-carrying strains reminds us to strengthen the management of antibiotic use and encourages the implementation of human feces surveillance, as well as actions to prevent and control the spread of multidrug resistant bacteria, especially bacteria resistant to carbapenems and colistin.