INTRODUCTION

Salmonella spp. are some of the most important agents that lead to enteritis in the world. Additionally, they can lead to more critical health problems such as bacteremia and enteric fever.¹ This group of bacteria include more than 2600 serotypes and consist of two species called S. enterica and S. bongori. S. enterica spp. are responsible for 99% of Salmonella infections and Salmonella Enteritidis and Salmonella Typhimurium are the most commonly isolated serotypes both in our country and in developed countries.² Virulence factors, which responsible for invasion, extraintestinal spread, and intracellular survival, are encoded by genes located in the Salmonella pathogenicity island (SPI). Although some pathogenicity islands seem to be preserved in Salmonella genus, others are unique for certain serotypes. Based on presence of SPI and SPI features, Salmonella serotypes differ from each other in terms of adaptation in host cells, virulence factors, and severity of infections.³

In most cases, resultant infections do not necessitate antibiotic treatment due to the self-limiting nature of disease. However, antibiotic treatment may be necessary for some situations such as invasive infections, advanced age, and immunosuppression.⁴ In such cases, ampicillin is widely used to treat Salmonella infections.⁵ Therefore, resistance to ampicillin has emerged and beta-lactam enzymes are primarily responsible of ampicillin resistance.⁶

The objectives of this study were to investigate beta-lactam resistance, the epidemiologic relationship, serotype distribution, and the prevalence of beta-lactamase genes, namely bla_TEM, bla_PER, bla_CTX-M, bla_SHV, and the virulence genes of clinical ampicillin-resistant S. enterica.

EXPERIMENTAL

Antimicrobial susceptibility

Salmonella enterica strains isolated in the Bacteriology Laboratory between 2010 and 2012 were used in our study. In vitro antibiotic susceptibilities of 117 S. enterica isolates were examined using modified Kirby-Bauer disc diffusion assays in accordance with Clinical and Laboratory Standards Institute (CLSI).⁷ Standard ampicillin (10 µg), cefotaxime (30 µg), ciprofloxacin (5 µg) sulfamethoxazole/trimethoprim (23, 75/1.25 µg) (Oxoid, United Kingdom) discs were used to detect resistance. The other part of the study was performed in ampicillin- resistant strains. Minimum inhibitory concentration (MIC) values of the isolates were also determined using broth microdilutions in accordance with the recommendations of the CLSI and S. enterica ATCC 04059 was used as a control strain.⁸

DNA isolation

DNA isolation was performed for use in polymerase chain reaction (PCR) studies. For this purpose, isolates were suspended and homogenized in 200 µL of sterile ultrapure water. The isolates were then incubated in a heat block at 95°C for 10 min. Microtubes were centrifuged at 13,000 rpm for 5 min. The supernatants were transferred to sterile microtubes and stored at -20°C for use in PCR studies.

Serotypes and epidemiologic relation

The strains were serotyped by the Turkish Public Health Agency, National Microbiology Reference Laboratory. The epidemiologic relations of the isolates were analyzed using PCR with enterobacterial repetitive intergenic consensus (ERIC)-2 and ERIC-1R primers.⁹ To evaluate similarity between these isolates, Jaccard coefficients were derived from the banding patterns. Dendrograms were constructed according to the unweighted pair group with arithmetic mean method, using Jaccard coefficients and MEGA software, version 4.0.

Beta-lactamase genes

bla_CTX-M, bla_TEM, bla_SHV, bla_PER genes were determined using primers targeting the relevant regions^10,11,12 using the conventional multiplex PCR method. PCR assays were run in 25 µL amplification mixtures composed of 5 µL bacterial DNA template, 2.5 µL Taq buffer, 1.5 mM MgCl₂, 200 µM dNTP, 30 pmol forward and reverse primers, and 1.25 U Taq polymerase.

Virulence genes

Six different virulence genes were analyzed in two different multiplex PCR reactions, using primers targeting the relevant genes.^13,14 For this purpose, a 5-µL bacterial DNA template, 2.5 µL Taq buffer, 1.5 mM MgCl₂, 200 µM dNTP, 20 pmol forward and reverse primers, 1.25 U Taq polymerase were prepared in 25 µL volume.

RESULTS

As a result of the disc diffusion test, ten (8.5%) out of 117 S. enterica isolates were detected as resistant to ampicillin. These resistant strains were susceptible to ciprofloxacin, cefotaxime, sulfamethoxazole/trimethoprim. The ampicillin MIC range of the isolates was found as 512-128 µg/mL. The strains were divided into seven different clusters based on ERIC-PCR results. The detected serotypes were as follows; S. Enteritidis (n=5), S. Infantis (n=2), S. Typhimurium (n=1), and S. Corvallis (n=1). One isolate could not be serotyped. Five strains involved bla_TEM genes, two strains contained bla_CTX-M genes. bla_PER and bla_SHV genes were not encountered. InvA, pipD, sopB, which are virulence genes, were detected in all strains, sifA in seven, pefA in four, and sopE in three strains.

DISCUSSION

The Salmonella genus comprises many members. Some Salmonella serotypes are known to be more commonly isolated. In light of this information and existing data; the most frequently isolated Salmonella serotype is known to be S. Enteritidis.¹⁵ In our study, five of the ampicillin resistant Salmonella strains were determined as S. Enteritidis. In 2014, Maraki and Papadakis⁴ determined S. Enteritidis as the most commonly isolated serotype of Salmonella (37.3%). In another study by Ozdemir and Acar,¹⁶ S. Enteritidis was determined as the most commonly isolated serotype from Salmonella isolates collected from 4 different provinces of Turkey. Our results show consistency with the literature.

Serotyping is the basic phenotypic method for epidemiologic investigation of isolates. Nonetheless, it cannot differentiate strains of the same serotype. Genotypic methods such as pulsed-field gel electrophoresis (PFGE), ERIC-PCR, and repetitive element palindromic PCR can distinguish the strains more effectively. Although PFGE is the gold standard method for fingerprinting, due to the lack of equipment and to avoid protocols lasting four-five days, a simpler method of ERIC-PCR was preferred.^17,18 In our study, strains were divided into 7 unrelated clusters using ERIC-PCR with an acceptable (≥0.90) discriminatory index value of 0.92. When the literature and data obtained in this study are evaluated, ERIC-PCR is considered to be a useful and easily applicable method for genotyping of strains.

Recently, Salmonella strains have shown resistance against many antibiotic groups. Ampicillin, which is a member of the beta-lactam antibiotics, is the first-line agent used in the treatment of Salmonella infections. Salmonella strains that are resistant to ampicillin and other beta-lactams pose a risk for public health.⁵ According to our disc diffusion results, ampicillin-resistant strains are susceptible to other groups of antibiotics such as ciprofloxacin, cefotaxime, and sulfamethoxazole/trimethoprim.

According to data gained abroad, the rate of ampicillin resistance against Salmonella varies from one country to another. As for studies abroad; in India, ampicillin resistance in S. enterica isolates was detected as 25% in 2011.¹⁹ It was 33% in Salmonella isolated from children in Cambodia,²⁰ 46% in Korea,²¹ 55% in Spain,²² and 8% in the United States.²³ In Turkey, there have been a few studies of clinical Salmonella strains that were isolated from children’s hospital. The rate of ampicillin resistance in Salmonella strains were determined as 25.8% in 2012,²⁴ and 19% in 2014.²⁵ However, resistance rates were higher than in our study. According to the our knowledge, the stress of starting antibiotic treatment empirically in pediatric patients, before culture results, might be responsible for higher ampicillin resistance rates than in our study.

In our study, five isolates with bla_TEM genes and two isolates with bla_CTX-M genes were found. bla_CTX-M-positive isolates were in the S. Ifantum serotype. Four of five bla_TEM gene positive isolates were in S. Enteritidis serotype, and the remaining strain was in S. Typhimurium. Although the most common beta-lactamase genes of Salmonella isolates are variants of bla_CTX-M and bla_SHV,²⁶ no bla_SHV gene was found in isolates used in our study. Different rates of beta-lactam resistance genes were reported in studies conducted abroad. Among 20 ampicillin-resistant Salmonella isolates, beta-lactamase genes were found to be bla_SHV 100%, bla_TEM 85%, bla_CTX-M 5% in one study;² and in 90 beta-lactam–resistant strains in Spain it was bla_TEM 22%, and bla_CTX-M 1%.²² In the Netherlands, a study with 34 Salmonella strains isolated from humans and the environment, Hasman et al.⁶ detected the bla_TEM gene in 19 (55%) strains, the bla_CTX-M gene in six (17%), and the bla_SHV gene in three (8%) strains; all 34 isolates were found to be resistant to penicillin. A study conducted on S. Typhimurium in 2011 in Turkey detected 23% bla_CTX-M gene, 76% bla_TEM gene, and 100% bla_SHV. The bla_PER gene could not be detected in any isolates²⁷.

Salmonella bacteria carry many different and complex virulence factors. Investigating the presence of virulence factors coded as different pathogenicity islands will guide us in the matter of discovering Salmonella pathogenesis. Our study is thought to be the first in Turkey to investigate the virulence factors of Salmonella, at least three virulence factors were detected in all strains. The invA gene was determined in all isolates involved in this study, and it was present independent from conditions such as serotype and resistance genes. Dione et al.²⁶ detected the invA gene in 99.5% of strains in a study. Another study conducted by Smith et al.⁵ in 2010 encountered the invA gene in all. The determination of high levels of the invA gene in different regions despite different serotypes and antimicrobial susceptibility profiles indicated the existence of a preserved region in this gene. Thus, the idea of using this gene for rapid diagnosis of Salmonella with PCR as a target region has arisen, and this idea has led to studies with positive outcomes.²⁸

Generally, the sopE gene, which has been shown to have the lowest prevalence, has been associated with epidemic cases.^26,29 In our study, the presence of three strains that involve this gene in the same group according to ERIC-PCR and their isolation in a short period of time indicated that they may have been isolated after a community- onset epidemic.

Hughes et al.¹⁴ detected pipD and sopB genes in all strains and these virulence factors have been associated with enteritis. The detection of these genes in all resistant isolates indicated a possible relationship between these virulence factors and resistance. A study conducted by Dione et al.²⁶ and our data gave similar results. Also, Khoo et al.³⁰ detected alterations in beta-lactam resistance as a result a mutations created in these two genes. Although our study did not directly show the relationship between resistance and virulence factors, it showed the necessity for further extensive studies about the relationship between these two factors.

The pefA gene, which was detected in four strains at the present study, may be located on the same or different plasmid with different virulence (spv) genes. Only a fraction of Salmonella serotypes carries different-sized plasmids, which are known as serovar specific. However, it is known that not every plasmid-carrying serotype includes the pefA gene. Therefore, it is thought that the pefA gene has a lower prevalence compared with other virulence factors.³¹ Hughes et al.¹⁴ showed that only three isolates out of 32 involved the pefA gene. Skyberg et al.¹³ performed a study on Salmonella of different serotypes, the pefA gene was found in 11 of 152 strains, and it was only present in serotypes of S. Typhimurium and S. Enteritidis. The low prevalence of the pefA gene in our study is compatible with the results of the other studies. Additionally, three out of four pefA gene-carrying strains belonged to the S. Enteritidis serotype, and this result is also consistent with the literature data.

The sifA gene, which enables the sustained vitality of Salmonella bacteria in macrophages, was detected in seven strains in our study. Hur et al.³² detected the sifA gene in all of 42 strains. Skyberg et al.¹³ determined the sifA gene in 137 out 158 isolates. The existence rate of the sifA gene in this study was similar to that of studies conducted abroad.

CONCLUSION

As a result, in light of studies both in our country and abroad, it is known that there are Salmonella strains that are resistant to antimicrobial agents, some of which are beta lactamase-producing. Further investigation on the resistance and virulence profiles of Salmonella strains will enable us to better understand the pathogenesis of infections and to be able to take better measurements and give proper treatments.

ACKNOWLEDGEMENTS

We are grateful to the UEPLA team for the serotyping of the strains as a surveillance program of the Public Health Institution of Turkey.

Conflict of Interest: No conflict of interest was declared by the authors.