The phenotypic and molecular characteristics of antimicrobial resistance of Salmonella enterica subsp. enterica serovar Typhimurium in Henan Province, China

Salmonella enterica subsp. enterica serovar Typhimurium is an important causative agent of human gastroenteritis and bacteremia in many countries. Worldwide, there are 93.8 million cases of human gastroenteritis due to Salmonella infection annually, associated with a death toll as high as 150,000 [1]. Over 2700 serovars have been identified using the White-Kauffmann-Le Minor scheme [1, 2], of which, S. Typhimurium is the most commonly associated with human and animal disease globally. S. Typhimurium is also the most prevalent serovars causing invasive Nontyphoidal Salmonella infections (iNTS), among iNTS cases, 63.7% occurred in children < 5 years of age globally [3]. Moreover, S. Typhimurium is the second most prevalent serotype in China [4].

Following the emergence of antimicrobial-resistant Salmonella, and in particular, the increasing prevalence of an epidemic multidrug-resistant (MDR) S. Typhimurium strain definitive phage type 104 (DT104) first observed during the 1990s [4], the antimicrobial resistance of Salmonella has become a matter of concern worldwide. Therefore, the appropriate selection of antimicrobial drugs in treating Salmonella infections is necessary. As previously described, cephalosporins (CEP) and ciprofloxacin (CIP) are first-line treatment agents for such infections [5], and azithromycin (AZI) has recently been approved by the US Food and Drug Administration as an additional therapy [6]. Salmonella isolates with reduced susceptibility to fluoroquinolones have been frequently reported in several countries, such as Kuwait, the United Arab Emirates, France [7‐9]. Moreover, strains resistant to third-generation CEP have been described [10]. Given that these drugs are also first-line treatments for Salmonella infections in children, for whom fluoroquinolones are contraindicated, this represents a troubling development [10, 11]. Furthermore, a study of Salmonella in Cambodia revealed high rates of decreased susceptibility to CIP and AZI [12]. Worryingly, a recent report also identified a number of strains with concurrent resistance to CEP, CIP, and AZI in China [13].

Within China, significant regional differences in bacterial antibiotic resistance profiles have been demonstrated [14]. Henan is the most populous province of Central China and a region of substantial labor productivity. High levels of migration are expected to accelerate the spread of drug-resistant bacteria, exacerbating the threat to public health. Therefore, the objectives of this study were to determine the antimicrobial resistance profiles and molecular epidemiological characteristics of S. Typhimurium present in Henan between 2006 and 2015. This investigation will assist in establishing a scientific basis for the prevention and control of intestinal infectious diseases and guide the selection of appropriate antimicrobials for treatment of S. Typhimurium infections.

S. Typhimurium is one of the most prevalent Salmonella serotypes in the world, which was frequently resistant to ampicillin, amoxicillin/clavulanic acid, ceftriaxone, chloramphenicol, kanamycin, nalidixic acid, streptomycin, trimethoprim-sulfamethoxazole, and tetracyclines [28‐30]. In this study, the S. Typhimurium isolates examined exhibited high rates of resistance to older-generation antimicrobials, and MDR (91.1%) showed the predominant resistance profile over the potential 10 year in Henan province, of which, 51.0% exhibited the ACSSuT resistance pattern. CEP, CIP, and AZI are recommended as first-line treatments for Salmonella infections [5, 6], and surprisingly, resistance rates to these antibiotics among the isolates tested were far higher than the values given in the 2014 National Antimicrobial Resistance Monitoring System (NARMS) report [27]. In addition, we found that the resistance rate of CEP and AZI showed a rise over time, while the resistance rate of CIP began to decline. Of concern, 91.1% of the isolates in the present investigation were MDR, an estimate higher than those in previous studies in Guangdong and Shanghai and in the abovementioned report [27, 31, 32]. More importantly, it is noteworthy that 13 (8.84%) isolates were co-resistant to CIP and CEP, and that all of these were also resistant to at least five additional antimicrobial classes, indicating that CIP and CEP resistant S. Typhimurium isolates have been disseminated among communities in Henan. Notably, three of the 13 isolates also exhibited resistance to AZI. Only some clinical S. Typhimurium strains have previously been documented with this resistance profile in China, 12 (2%) of 546 S. Typhimurium isolates resistant to both CIP and AXO were recovered from patients in hospitals during the period of 2005 to 2011; among these 12 isolates, two were also resistant to AZI [13]. And ACSSuT Salmonella co-resistant to quinolones and cephalosporins will make treatment even more difficult, and the spread of these isolates will pose a real threat to global public health. The above analyses therefore reveal that S. Typhimurium resistance patterns have obviously changed in recent years (Table 2 and Table S1), and empirical therapy should keep pace with these changes. Of concern is 6 (4.1%) strains are resistant to all antibiotic classes tested in our study, it meant we were almost impossible to find an effective treatment for infections. This situation demands regular surveillance of antimicrobial resistance and implements an efficient infection control program. We should control the injudicious use of antibiotics, and manage the antibiotics which are readily available in pharmacies without a prescription. Beacause antibiotic resistance could be driven by antibiotic consumption, the changing composition of consumption may also reflect alterations in patterns of resistance [33]. Otherwise, antimicrobial resistance will become more and more serious with such a sustained selective pressure. Henan Province is located in Central China, representing approximately 7% of the Chinese population, and is a major transport hub with substantial population mobility. If these MDR strains identified here become as prevalent as definitive phage type 104 worldwide, a more serious threat to national and international public health would be posed. It is therefore essential that antimicrobial resistance be monitored and appropriate drugs be chosen for S. Typhimurium infections.

In order to facilitate understanding of the fundamental factors underlying bacterial antimicrobial resistance and establish measures for its prevention, examination of the molecular mechanisms responsible is urgently needed. Salmonella CIP resistance has principally been attributed to point mutations in the quinolone resistance-determining regions of genes encoding the target gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) enzymes [7, 34]. Two gyrA mutations affecting amino acid residues 83 and 87 have been identified in S. Typhimurium with high-level resistance to CIP, and variations in other target enzyme-encoding genes such as parC and parE increase such resistance [35, 36]. In the present study, 43 CIP-resistant isolates were identified, all of which had at least one gyrA mutation. Thirty-nine (91%) of these carried two such mutations and showed high-level resistance. One isolate exhibiting a high degree of CIP resistance carried two gyrA mutations (Ser83Phe and Asp87Gly), one parC mutation (Ser80Arg), and a single parE mutation (Ser458Pro). This parE sequence variation has been reported previously in Taiwan, Hong Kong, and Wuhan [37‐39], suggesting that it has the potential to become prevalent in China. As previously described, PMQR genes such as qnr and aac(6′)-Ib-cr have also been established as conferring CIP resistance [40]. The first PMQR gene was identified in a clinical isolate of Klebsiella pneumoniae in 1998 [41], and to date, various PMQR gene types have been detected in clinical Salmonella isolates from humans worldwide [42‐44]. In this study, the genes qnrS1 and aac(6′)-Ib-cr were found to be present in 25.6% (11 isolates) and 16.3% (seven isolates) of the 43 CIP-resistant isolates, respectively. Interestingly, 12 isolates simultaneously carried two gyrA mutations, a single parC mutation, and at least one PMQR gene, and thus demonstrated a high level of CIP resistance. However, three isolates highly resistant to CIP harbored only single gyrA mutations affecting amino acid position 87. The fact that they also carried qnrS1 or aac(6′)-Ib-cr indicates that the presence of PMQR genes can increase CIP MICs [37, 40].

Production of β-lactamases is considered the predominant mechanism of bacterial resistance to CEP [45]. Among the 32 CEP-resistant isolates in the current work, bla_OXA was the most frequently observed β-lactamase gene (n = 28), with only bla_OXA-1 being detected, suggesting that it is prevalent among S. Typhimurium in Henan. OXA-type β-lactamases are characterized by high levels of hydrolytic activity against oxacillin and cloxacillin, and confer resistance to AMP and CEP [46], compounding the difficulty faced in choosing antimicrobials for treatment of S. Typhimurium infections. In recent years, the bla_OXA gene has also been recorded at high frequencies among other Enterobacteriaceae [47]. Previous studies have shown that CMY-2-type β-lactamases encoded by the plasmid-borne bla_CMY-2 gene are the most prevalent and problematic damaging β-lactamase of such enzymes [48]. Although the bla_CMY-2 resistance gene has been documented in Salmonella isolates from many countries [49], its presence in China has only been reported in Shandong, Shanxi, and Sichuan Provinces [50‐52]. In the current study, one isolate was positive for bla_CMY-2, bla_CTX-M, and bla_OXA and resistant to all antimicrobial classes tested. To the best of our knowledge, this is the first report of bla_CMY-2-positive Salmonella in Henan. Of the 32 CEP-resistant isolates in our investigation, 40% harbored bla_CTX-M genes, while 28.1% carried the bla_TEM-1 resistance gene. We also found that some of these isolates harbored at least two CEP-resistance genes at the same time. As ESBL genes are usually located on antimicrobial-resistance plasmids, they can be easily transferred between different species of bacteria [53]. We should therefore pay consideration to such phenomena and make every effort to take preventive measures.

ST19 and ST34 have been shown to be common S. Typhimurium STs responsible for infections worldwide [54‐56]. Here, these two STs were also the most commonly encountered, indicating their predominance in Henan. Of the 43 CIP-resistant isolates, 39 were categorized as ST19, indicating a relationship between this ST and resistance to CIP (P < 0.05). Moreover, 13 MDR isolates co-resistant to CIP and CEP were identified as ST19, suggesting that the ST19 is prevalent among MDR S. Typhimurium in Henan. According to our data, we note that only by combining etiological and epidemiological information can the characteristics of the spread of resistant clonal strains be better understood.

We analyzed the antimicrobial resistance and basic molecular mechanisms underlying CIP and CEP resistance of S. Typhimurium isolates in Henan, China, and explored their genetic relatedness. These isolates not only exhibited high rates of resistance to traditional antimicrobials but also show high resistance rates to the first-line treatments for Salmonella infection. More importantly, we identified certain MDR isolates co-resistant to CEP, CIP, and AZI, suggesting that the choice of treatment for Salmonella infection has become increasingly difficult. Among the isolates, we detected various plasmid-encoded antimicrobial-resistance genes, including PMQR, ESBL, and mphA genes, with some isolates even carrying two or more types, posing a serious threat to global public health. Therefore, more comprehensive surveillance is essential to prevent further spread of resistant clonal strains.