Research Article: DNA Sequence Analysis of Plasmids from Multidrug Resistant Salmonella enterica Serotype Heidelberg Isolates

Date Published: December 10, 2012

Publisher: Public Library of Science

Author(s): Jing Han, Aaron M. Lynne, Donna E. David, Hailin Tang, Joshua Xu, Rajesh Nayak, Pravin Kaldhone, Catherine M. Logue, Steven L. Foley, Mark Alexander Webber.


Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc) A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

Partial Text

Salmonella enterica is a major cause of foodborne illnesses. In the United States alone, over 1 million cases of salmonellosis are predicted to occur annually [1]. The economic cost associated with these infections has been estimated to be between 2.3-billion to 9.4-billion US dollars each year due to loss of work, medical care, quality of life and death associated with salmonellosis [2]–[4]. Among the over 2,500 serovars of S. enterica identified, serovar Heidelberg is among the top five most commonly identified serotypes in human salmonellosis and is commonly detected among Salmonella isolates submitted to the National Veterinary Services Laboratory that originated from poultry and swine [5]. Annually, infections with S. Heidelberg lead to approximately 84,000 cases of salmonellosis and contribute to approximately 7% of the Salmonella-related deaths in the U.S.; the second highest percentage after S. Typhimurium [6], [7]. Poultry products such as turkey, chicken and eggs are important sources of S. Heidelberg infections in humans [7]. Contamination of poultry products with Salmonella remains an important concern because of consumer preferences for poultry products. Data from the U.S. Department of Agriculture (USDA) indicate nearly four-fold increase in the per capita consumption of poultry products in the U.S. over the past half century [8].

DNA sequencing of plasmids from five multidrug resistant S. enterica serovar Heidelberg isolates showed that each isolate studied had at least one smaller plasmid of less than 10 kb in size (all without antimicrobial resistance genes) along with larger plasmids greater than 100 kb (Table 1). This study focused on the sequencing results and characterization of those plasmids containing antimicrobial resistance determinants. Four of the five isolates in the study harbored two plasmids containing antimicrobial resistance genes that were consistent with the observed susceptibility profiles for the isolates. With the exception of isolates 163 and 696, the plasmids from the isolates were quite different from each other. Isolate 163, which originated from a turkey diagnostic specimen, contained resistance plasmids of approximately 135 and 121 kb in size, as well as a 34 kb plasmid carrying genes associated with VirB/D4 Type IV Secretion System (T4SS; pSH163_34, accession number JX258656) and two small 3.4 and 3.3 kb plasmids that are very similar to CP001119 and CP001149, respectively. Similarly, isolate 696 which was collected from a turkey processing plant contained resistance plasmids of 135 and 117 kb and the VirB/D4 T4SS (pSH696_34, JX258654), 3.4 and 3.3 kb plasmids similar to isolate 163. The 135 kb plasmids from isolates 163 (pSH163_135; accession number JN983045) and 696 (pSH696_135; JN983048) were members of the IncA/C type and clustered most closely to one another in comparison to previously sequenced IncA/C-like plasmids (Figure 1). The plasmids share similar sets and arrangement of genes in their genetic backbone and carry the same sets of antimicrobial resistance (Table 1) and disinfectant resistance genes, including those associated with mercury and quaternary ammonium compound resistance (Figure 1). Both isolates carried an additional resistance plasmid, which are IncFIB plasmids of approximately 120 kb (isolate 163; pSH163_120; JN983046) and 117 kb (isolate 696; pSH696_117; JN983047) in size (Figure 2). Each plasmid contained genes associated with resistance to kanamycin (Kan), streptomycin (Str), sulfonamides (Sul) and tetracyclines (Tet; Table 1). These IncFIB plasmids also contained multiple iron acquisition genes, including those of the aerobactin (iucABCD and iutA) and the Sit iron transport (sitABCD) operons (Figure 2).

This study highlights the sequencing results of antimicrobial resistance plasmids from five multidrug resistant S. Heidelberg strains isolated from food animal and human sources. The three fully sequenced IncA/C plasmids in the study shared sequence similarities with a number of other plasmids that were previously sequenced, however they had unique characteristics. There are more than 20 IncA/C plasmids that have been fully sequenced and archived in GenBank as of 8/15/2012, of these only two (JF503991 and JF714412) grouped together with near identical similarity. Thus these multidrug resistance plasmids, which share some common backbone sequences, are genetically diverse. Interestingly, pSH163_135 and pSH696_135 were closely related to each other; but they were distinct from many of the other IncA/C plasmids, such that portions of the transfer-associated regions, Tra 1 and 2, were missing compared to other IncA/C plasmids using Mauve. Some of the genes that were not present included traNUW, ssb, bet and multiple genes encoding hypothetical proteins [28]. These findings were consistent with previous results that showed multiple Salmonella isolates, including a S. Heidelberg isolated from a turkey, appeared to lack the portions of the transfer region in their IncA/C plasmids [19]. In addition, the Tn21 regions of pSH163_135 and pSH696_135, containing blaTEM, aadB and cmlA, were unique compared to other IncA/C plasmids [19], [29], but were similar to mobile elements in overlapping regions of plasmids from two Klebsiella pneumoniae plasmids (AY123253 and AJ704863).