Tracing the source of blaCTX-M-15 producing ST34 Salmonella typhimurium isolated from feces and wound secretion

Background Salmonella enterica serovar typhimurium infection is common in food-borne diseases, isolating it from surgical incisions is rare. The aim of this study was to trace the transmission source of a surgical incision infected with Salmonella typhimurium in a Yunnan Province hospital patient and elucidate the underlying molecular mechanisms of antibiotic resistance. Methods Primers were designed to amplify the drug resistant genes by polymerase chain reaction (PCR). Susceptibility to antibiotics was determined using Etest. The identical macrorestriction proles were analyzed by pulsed eld gel electrophoresis (PFGE) using XbaI. The two isolates were characterized using an agglutination test and multilocus sequence typing (MLST). Results The MLST analysis revealed that isolates SM043 and SM080 belonged to the same genotype ST34, and the PFGE revealed that SM043 and SM080 had high similarity. The two S. typhimurium strains SM043 and SM080 were resistant to third generation cephalosporins. SM043 harbored blaCTX-M-15 , blaTEM-1 , qnrS-1 , qnrB , and acc-3 resistance genes, while blaCTX-M-15 , blaTEM-1 , blaCMY-2 , qnrS-1 , and acc-3 were detected in SM080.


Background
Salmonella is a member of the Enterobacteriaceae family, which was originally characterized by the ability of the bacteria to metabolize citrate as a sole carbon source [1]. Members of the genus Salmonella cause a wellcharacterized spectrum of disease in humans, which range from asymptomatic carriage to fatal typhoid fever. Third generation cephalosporins are commonly used as rst-line antibiotics to treat invasive infections or enteric fevers caused by Salmonella spp. due to their pharmacodynamic properties and the low prevalence of resistance [2,3]. Our previous study found that the drug resistance rate to third generation cephalosporins was low in the Yunnan Province [4]. Therefore, the resistance mechanism of Salmonella to third-generation cephalosporins in this region is still not available. However, outbreaks or cases of infection caused by Salmonella resistance to extended-spectrum cephalosporins have been reported around the world [5][6][7]. Recently, the growing antibiotic resistance of Salmonella enterica serovar typhimurium is creating new challenges for the control and prevention of potentially lethal infections [8].
Salmonella typhimurium was rst isolated in cattle in 1988; the infections caused by it are more common in foodborne diseases. In China, S. typhimurium was reported as the most frequently isolated serotype of nontyphoidal Salmonella from foodborne illnesses [9,10]. Salmonella infections involving sepsis, liver abscesses, surgical wounds, joint infections, and infected burn wounds have also been described in case reports from around world [9,11]. In 2018, two S. typhimurium strains resistant to 3rd generation cephalosporins were isolated from a teaching hospital in Yunnan Province, China. (The name of the hospital has been concealed to maintain anonymity). One of the isolates was from a rare surgical incision secretion. In this study, we aimed to determine the source of the S. typhimurium to prevent the salmonellosis from spreading or causing an outbreak. The antimicrobial resistance pro les of two S. typhimurium were analyzed simultaneously to elucidate the molecular type and drug resistance mechanisms.

Bacterial isolates
Two S. typhimurium strains, SM043 and SM080, resistant to third-generation cephalosporins were isolated from clinical specimens in 2018. SM043 was isolated from the feces of a 9-year-old patient with diarrhea and fever in October 2018. In November 2018, SM080 was isolated from an infected incision secretion from a 4year-old child after surgery due to a car accident. The two isolates were identi ed by the VITEK 2 system (bioMérieux, Lyon, France). Serotypes were detected using the White-Kauffmann-Le Minor (WKL) scheme based on serological detection; the diagnostic serum of Salmonella (STATENS SERUM INSTITUTE, Denmark) was used to determine the serotype.

Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined by the disk diffusion method and the automated VITEK 2 Compact system with Gram-negative bacteria cards (bioMérieux, Lyon, France). The Etest method was employed to verify the drug sensitivity of 5 antibiotics, and the results were interpreted based on the CLSI guidelines [12] [13]. Escherichia coli (ATCC 25922) was used as the quality control strain for antimicrobial susceptibility testing.

Detection of drug-resistant genes
Bacterial chromosomal DNA was obtained with a TIANamp Bacterial DNA Kit according to the manufacturer's instructions (TIANGEN BIOTECH, Beijing, China). PCR and DNA sequence analysis were performed to con rm the presence of drug-resistant genes. The primers used in this study were described previously [14][15][16][17][18][19][20]. The βlactamase genes included Ambler class A (bla CTX−M , bla TEM , bla SHV , bla KPC , and bla GES ), class B (bla NDM ), class C (bla CMY , bla ACT , and bla DHA ), and class D (bla OXA−48 ). Moreover, genes related to quinolone activity included qnrA, qnrB,and qnrS, and the aac gene were also detected. All amplicon sequences were compared with those in the GenBank nucleotide database (www.ncbi.nlm.nih.gov/blast/).

Molecular typing
The SM043 and SM080 strains were genotyped using MLST and PFGE. Seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, thrA) were ampli ed according to the protocol described on the MLST website (https://pubmlst.org). Genotyping was carried out by referring to the molecular typing method of Salmonella serotype pulse eld gel electrophoresis (PFGE) in PulsenetChina. Salmonella was entrapped with SeaKem Gold Agarose and digested with XbaI. The DNA fragment obtained was classi ed into PFGE by Chefmapper. Finally, Cluster analysis was performed with Bionumerics 7.6.

Antimicrobial susceptibility testing
The drug-resistance pro les of SM043 and SM080 were consistent; the 3rd -and 4th -generation cephalosporins including ceftriaxone, cefotaxime, and cefepime showed high resistance. The MIC values of aminoglycoside and tetracycline were also high. The two isolates were sensitive to β-lactam compound drugs, carbapenems, macrolides, and quinolones. These results are summarized in Table 1.

Gene type
MLST analysis showed that SM043 and SM080 were de ned as the single sequence type ST34. The two strains were successfully typed by PFGE and were classi ed into same PFGE cluster (Fig. 1.)

Discussion
While Salmonella enterica serovar typhimurium infection is common in food-borne diseases, isolating it from surgical incisions is rare. There were two S. typhimurium isolates from a hospital in the Yunnan Province in 2018, one from an outpatient and one from an inpatient. The rst case, SM043, was isolated from fecal specimens from a pediatric outpatient with diarrhea in October 2018. The second case, SM080, was isolated from a child who developed a surgical wound infection after emergency trauma surgery in November 2018.
PFGE analysis of the two Salmonella typhimurium isolates exhibited an identical macrorestriction pro le. Further, MLST analysis de ned the two S. typhimurium strains as the single sequence type ST34. Based on the genotype results of PFGE and MLST analysis, the genetic homology of the two isolates was high.
Environmental swabs from the bed linens, stethoscopes, doorknobs, and the hand swabs obtained from doctors and nurses, were collected and tested. However, S. typhimurium was not isolated from any of the environmental surfaces. According to the eld investigation and analysis, we know that both the trauma surgery emergency department and pediatric outpatient department of the hospital were located in a temporary building due to construction in the hospital. Both children were con rmed patients in the temporary hospital building. Healthcare-associated infections (HAIs) are known be key causes for morbidity and mortality in hospitalized patients [21]. Previous studies have suggested that environmental contamination plays a signi cant role in HAIs and in the unrecognized transmission of pathogens [22]. Cross-transmission of these pathogens can occur via the hands of healthcare workers, who become contaminated directly from contact with patients or indirectly by touching contaminated environmental surfaces. S. typhimurium has increased levels of resistance to stress conditions due to various survival mechanisms; this enables it to survive for a prolonged period of time in harsh environments [23]. Unfortunately, in this study, S. typhimurium could not be isolated from the hospital environments. In an earlier report of the Salmonella outbreak [24], epidemiological investigation revealed that the probable source of this outbreak was leakage of sewage water in the water supply system; however, no pathogens were isolated from the water samples. Salmonella typhimurium can be present in the environment for a long time, and several studies have shown that microbial contamination of the ward environment can be a source of nosocomial infection transmission.
According to the eld investigation, we know that due to partial renovation and construction, the hospital's the emergency department of trauma surgery and pediatric outpatient department were relocated to a temporary building. Closer investigation and understanding of the building structure indicate that the distribution of the clinic wards and the allocation of resources in the temporary location were not su cient in terms of complying with the prevention measures during the transitional period. First and foremost, the lack of alcohol hand sanitizers, soaps and tissues in the ward has negatively impacted the basic hand hygiene practices of patients and their families, as well as the compliance of medical personnel in the wards. In this study, surgical incision infections occurred in children as young as 4 years old, who were primarily cared for by family members. Secondly, the building layout is not reasonable, especially toilet con guration is signi cantly insu cient. The number of toilets in this transition site, which is an old building, is low and the space is very limited, forcing medical staff, patients and patients' families to share toilets. The utilization rate of these toilets is extremely high. Due to the lack or absence of hand sanitizers in toilets, people cannot wash their hands after using toilets. Thirdly, we also found that the janitor team only regularly clean and disinfect the wards, ward grounds, but no beds in the wards, door handles, railings, light switches, window edges, toilet door handles, and other environmental surfaces were being effectively disinfected, which likely contributed to the spread of bacteria in the environment. The hands of health care workers are the main source of pathogen transmission in hospitals. It has been reported that 20-40% of nosocomial infections are caused by microorganisms present in the hospital environment that are transmitted directly or through the hands of healthcare workers (HCW). Therefore, in this research, we speculate that S. typhimurium exists in the hospital environments, which serve as potential reservoirs to pose a serious threat to patients. The surgical incision infection caused by S. typhimurium SM080 is most likely caused by person-to-person transmission. This may be due to deteriorating hand-hygiene, poor infection control measures, and crowded hospital wards. The nosocomial infection in the emergency trauma surgery was nally controlled by the following intervention measures. Highlighting as the most important intervention is the uninterrupted provision of hand-washing consumables and improved hand-washing facilities. Second, hand hygiene and nosocomial contact prevention education for patients and staff. Third, regular trainings for the ward custodian team, strengthening the cleaning and disinfecting practices within the ward environment. As of present time, no other nosocomial infections caused by salmonella were reported.
In China, cephalosporins and uoroquinolones are commonly used to treat salmonellosis. Resistance to thirdgeneration cephalosporins is increasing in Salmonella spp. This is mainly due to the production of acquired AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) [25]. By harboring at least one ESBL or AmpC gene, both SM043 and SM080 were resistant to 3rd -and 4th -generation cephalosporins. It is important to continuously monitor the antimicrobial resistance and genetic status in S. typhimurium in order to detect emerging resistance trends. This is the rst time that bla TEM−1 , bla CTX−M−15 , and bla CMY−2 have been detected in S. typhimurium in the Yunnan province.

Conclusions
In summary, contaminated hospital environments can serve as potential reservoirs for pathogens such as S. typhimurium. S. typhimurium is spread not only by the fecal-oral route, but also by contact with contaminated hospital environments. Our study can help people by increasing awareness regarding the potential risks of S. The authors declare no con ict of interest. Availability of data and materials The datasets used and/or analyzed in this study are available from the corresponding author on reasonable request. Pulsed-eld gel electrophoresis patterns of XbaI-digested total DNA of two Salmonella typhimurium isolates.