Antimicrobial resistance, presence of integrons and biofilm formation of Salmonella Pullorum isolates from eastern China (1962–2010)

Abstract

Three hundred and thirty-seven isolates of Salmonella Pullorum from eastern China between 1962 and 2010 were characterized for antimicrobial susceptibility (disk diffusion method), the presence of integrons (polymerase chain reaction followed by sequencing) and the ability to form biofilms (semi-quantitative adherence assay). Two hundred and fifty-eight isolates (76.6%) exhibited multiple drug resistance (MDR; resistant to at least three different classes of antimicrobials), and the level of drug resistance is increasing with time. There were three isolates (9.4%) exhibiting MDR from 1962 to 1968. MDR rates began to increase for isolates between 1970 to 1979 and 1980 to 1987 (64.6 to 78.7%). The MDR rates reached 96.6% for isolates between 1990 and 2010. Polymerase chain reaction screening for integrons showed that 75 isolates (22.3%) were positive for class 1 integrons while none were positive for class 2 integrons. All of the class 1 integron-positive isolates exhibited MDR and were more frequently resistant than the negative isolates. Two hundred and twenty isolates (65.3%) had the ability to form biofilms, and bacterial resistance levels to cefamandole, trimethoprim and trimethoprim/sulfamethoxazole were significantly higher for biofilm-positive groups than the biofilm-negative groups. Our data show that multidrug resistance is common among S. Pullorum isolated from eastern China, being more frequent after 1990 than before 1990, and the presence of class 1 integrons is associated with multidrug resistance.

Introduction

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the causative agent of pullorum disease in poultry, an acute systemic disease that results in a high mortality in young chicks (Shivaprasad, 2000). Because of its vertical and horizontal transmission, S. Pullorum is capable of causing great economic loss in the poultry industry. Although pullorum disease is well controlled in many developed countries, it is still common in many parts of the world (Barrow & Freitas Neto, 2011). Antimicrobials have played an important role in the control of Salmonella, but misuse of antimicrobials has resulted in the evolution of multidrug-resistant strains, which makes prevention and treatment more difficult. The increasing appearance of multidrug-resistant Salmonella has become a worldwide concern. The link between the use of antimicrobials in food animals and the emergence of antimicrobial resistance of human pathogenic bacteria is well documented (Martínez & Baquero, 2002; Ahmed et al., 2009; Araque, 2009). Integrons are capable of capturing and mobilizing antimicrobial resistance bacterial genes. Class 1 and class 2 integrons are the most common integron types identified in Salmonella and play an important role in the dissemination of resistance genes among these pathogens (Orman et al., 2002; Antunes et al., 2006; Khemtong & Chuanchuen, 2008). Likewise, biofilms are important factors in antimicrobial resistance, and play a key role in the pathogenesis of many bacterial infections. Bacteria with biofilms are inherently protected from their surrounding environment and often exhibit increased resistance to host defence and antimicrobial agents, making these infections difficult or impossible to eradicate (Costerton et al., 1999; Hall-Stoodley et al., 2004). In the study described herein, antimicrobial susceptibility profiles, the prevalence of class 1 and class 2 integrons and the ability to form biofilms were examined in a collection of S. Pullorum strains isolated from 1962 to 2010 in eastern China. This study will help to understand the tendency of S. Pullorum to become resistant to antimicrobials with time, analyse antimicrobial resistance and correlate resistance with integrons and biofilms. Such information should prove useful in formulating guidelines for appropriate antimicrobial usage.

Materials and Methods

S. Pullorum isolates

Three hundred and thirty-seven S. Pullorum isolates were evaluated in this study. All of the isolates were recovered from chickens with clinical signs of pullorum disease. All bacterial strains were isolated from the heart, lung, liver, spleen, bile, testis and ovary samples in five different provinces in eastern China from 1962 to 2010: Jiangsu, Shanghai, Shandong, Anhui and Zhejiang. Most of the samples were obtained from individual cases in commercial stock. Each sample was plated onto MacConkey agar (Oxoid, Basingstoke, UK) plates and incubated at 37°C for 24 to 36 h. S. Pullorum isolates were identified biochemically using the Automatic Microbiological Analysis Manufacturer (Biolog, Hayward, California, USA) and serotyping was performed using standard agglutination tests with O and H antisera (Tianrun, Ningbo, China). The isolates were archived at −80°C in tryptic soy broth (Oxoid) containing 20% glycerol. The background and history of each isolate was recorded for further analysis and comparison.

Antimicrobial susceptibility testing

The antimicrobial susceptibilities of S. Pullorum isolates were determined using the disk diffusion method on Muller Hinton agar (Oxoid) standardized by the Clinical and Laboratory Standards Institute guidelines (CLSI, 2012). The isolates were tested against a panel of 16 antimicrobial agents (Oxoid): ampicillin (10 µg), carbenicillin (100 µg), cefamandole (30 µg), cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), nitrofurantoin (100 µg), spectinomycin (100 µg), streptomycin (10 µg), sulfamethoxazole (300 µg), tetracycline (30 µg), trimethoprim (5 µg), and trimethoprim/sulfamethoxazole (1.25/23.75 µg). Results were obtained after incubating samples for 16 to 18 h at 37°C. Escherichia coli (ATCC25922), Enterococcus faecalis (ATCC29212), and Staphylococcus aureus (ATCC29213) were quality control organisms.

Detection of class 1 and class 2 integrons

The primers to amplify class 1 and class 2 integrase genes in clinical specimens are designed as previously described (Lévesque et al. 1995; Goldstein et al., 2001). Polymerase chain reaction (PCR) cycling conditions were initial denaturation at 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 55/50°C for 30 sec, extension at 72°C for 1 min followed by final extension at 72°C for 10 min.

PCR products were purified using the Quick Gel Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Sequencing was carried out by the DNA Synthesis and Sequencing Facility at Sangon Biotech Co., Ltd (Shanghai, China), and compared with the data in GenBank using BLAST to confirm homology.

Biofilm formation of the isolates

Biofilm formation for all 337 isolates was assessed using the semi-quantitative adherence assay (Stepanović et al. 2004; Peeters et al., 2008). The optical density (OD) obtained at 630 nm using a Sunrise Absorbance Reader (PerkinElmer, Waltham, Massachusetts, USA). All tests were carried out in triplicate. OD values from the wells that had not been inoculated with bacteria were used as negative controls. The cutoff value (ODc) for determining biofilm production was defined as two times the negative control value, OD > ODc was considered positive and OD ≤ ODc considered negative (Yi et al., 2004).

Data analysis

The isolates were classified as susceptible or resistant according to the zone diameter interpretative standard recommendations by CLSI (2012). To test for significance in the difference of resistance rate overall, the chi-square test was performed using the Statistical Package for the Social Sciences (SPSS version 13.0; Chicago, Illinois, USA).

Results

Antimicrobial resistance of S. Pullorum isolates

Three hundred and twenty-five isolates (96.4%) showed resistance to at least one antimicrobial and none was resistant to all 16 antimicrobials (Table 1). The isolates exhibited the highest frequencies of resistance to trimethoprim, streptomycin, tetracycline and sulfamethoxazole, while lower rates were observed for gentamicin, ciprofloxacin, chloramphenicol, kanamycin and cefotaxime. The resistance rates of the isolates to antimicrobials increased significantly (P<0.01) over the last 50 years. The isolates from the 1960s had low resistance rates. The resistance rates in the isolates from the 1970s began to increase, with resistance to trimethoprim/sulfamethoxazole and spectinomycin increasing rapidly. Resistance rates of the isolates from the 1980s continued to increase, and isolates resistant to kanamycin and gentamicin appeared. In the isolates from the 1990s, resistance rates reached their highest level with streptomycin, tetracycline, ampicillin, carbenicillin and nitrofurantoin. Isolates resistant to nalidixic acid and chloramphenicol appeared during this time period. For the isolates from 2000 to 2010, some antimicrobial resistance rates began to ebb but remained at high levels. Isolates resistant to cefotaxime and ciprofloxacin appeared during this interval.

Table 1.  Antimicrobial resistance of S. Pullorum isolates.

	Number of resistant isolates
Antimicrobial	1962 to 1968 (n=32)	1970 to 1979 (n=82)	1980 to 1987 (n=75)	1990 to 1999 (n=56)	2000 to 2010 (n=92)	Total (n=337)
Ampicillin	2	16	19	39	40	116
Carbenicillin	3	6	11	28	38	86
Cefamandole	6	27	32	29	63	157
Cefotaxime	0	0	0	0	8	8
Chloramphenicol	0	0	0	6	8	14
Ciprofloxacin	0	0	0	0	15	15
Gentamicin	0	0	2	4	12	18
Kanamycin	0	0	1	2	10	13
Nalidixic acid	0	0	0	3	61	64
Nitrofurantoin	2	19	24	24	20	89
Spectinomycin	0	4	10	13	18	45
Streptomycin	5	26	49	53	75	208
Sulfamethoxazole	10	66	46	25	31	178
Tetracycline	6	35	53	49	55	198
Trimethoprim	3	68	70	52	86	279
Trimethoprim/sulfamethoxazole	0	20	33	35	79	167

Of the 337 isolates tested, 258 (76.6%) showed multiple drug resistance (MDR; resistant to at least three different classes of antimicrobials). The isolates showed an increasing tendency towards MDR with time (Table 2). There were only three isolates that exhibited MDR from 1962 to 1968. MDR began to increase between 1970 and 1979, when most of the isolates were resistant to four or five antimicrobials (52.5%). The rates continued to rise and most of the isolates exhibited resistance to five or six antimicrobials in 1980 to 1987 (50.7%). The MDR rates were very high between 1990 and 2010, and nearly 100% of the isolates exhibited resistance to three or more antimicrobials. Most of the isolates were resistant to six or seven antimicrobials (43.9%). Twelve isolates were resistant to 10 or more antimicrobials.

Table 2.  Multidrug resistance of S. Pullorum isolates.

	Number of resistant isolates
Number of resistant antimicrobials	1962 to 1968 (n=32)	1970 to 1979 (n=82)	1980 to 1987 (n=75)	1990 to 1999 (n=56)	2000 to 2010 (n=92)	Total (n=337)
1	13	6	1	0	0	20
2	7	10	6	0	2	25
3	3	15	11	3	4	36
4	1	24	12	5	7	49
5	0	19	23	9	9	60
6	0	3	15	13	19	50
7	0	1	5	10	23	39
8	0	0	2	9	12	23
9	0	0	0	3	8	11
10	0	0	0	3	3	6
11	0	0	0	1	3	4
12	0	0	0	0	2	2

Association of the presence of class 1 integrons with antimicrobial resistance

Of the 337 S. Pullorum isolates tested, 22.3% carried int1 but none of the isolates were positive for int2. The int1 genes were detected in 59.8% and 35.7% of isolates from 1990 to 1999 and 2000 to 2010 respectively (Table 3), while the isolates from 1962 to 1987 were all negative. The resistance rates of int1(+) isolates were higher than those that were int1(−) (Table 4). The differences in int1(+) and int1(−) resistance rates to ampicillin, carbenicillin, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, sulfamethoxazole, spectinomycin, tetracycline, trimethoprim and trimethoprim/sulfamethoxazole were significant (P<0.05), while resistance rates to cefamandole, cefotaxime, gentamicin, kanamycin and nitrofurantoin were not significant (P>0.05). All S. Pullorum int1(+) isolates were MDR, while the negative isolates were 69.8% MDR (P<0.001).

Table 3.  Class 1 integrons and biofilm formation of S. Pullorum isolates.

Years	Number of isolates	Number of class 1 integron-positive isolates	Number of biofilm-positive isolates
1962 to 1968	32	0	16
1970 to 1979	82	0	47
1980 to 1987	75	0	46
1990 to 1999	56	20	40
2000 to 2010	92	55	71
Total	337	75	220

Table 4.  Correlation between class 1 integrons and antimicrobial resistance.

	Number of resistant isolates
Antimicrobial	Positive (n=75)	Negative (n=262)	P value
Ampicillin	34	82	0.024
Carbenicillin	27	59	0.018
Cefamandole	40	117	NS
Cefotaxime	3	5	NS
Chloramphenicol	8	6	0.001
Ciprofloxacin	7	8	0.020
Gentamicin	6	12	NS
Kanamycin	4	9	NS
Nalidixic acid	23	41	0.003
Nitrofurantoin	25	64	NS
Spectinomycin	16	29	0.021
Streptomycin	60	148	<0.001
Sulfamethoxazole	51	127	0.003
Tetracycline	53	145	0.017
Trimethoprim	69	210	0.017
Trimethoprim/sulfamethoxazole	49	118	0.002
MDR	75	183	<0.001
NS, not significant.

Relationship between biofilms formation and antimicrobial resistance

There were 65.3% isolates with the ability to form biofilms (OD > ODc = 0.146). The proportion of biofilm-positive isolates increased over time (Table 3). The antimicrobial resistance rates of positive isolates were higher than those of negative isolates (Table 5), but the differences were not significant except for cefamandole, trimethoprim and trimethoprim/sulfamethoxazole (P<0.05). The proportion of MDR for positive and negative isolates was 79.5% and 70.9%, respectively (not significantly different).

Table 5.  The correlation between biofilms and antimicrobial resistance.

	Number of resistance isolates
Antimicrobial	Positive (n=220)	Negative (n=117)	P value
Ampicillin	79	37	NS
Carbenicillin	60	26	NS
Cefamandole	112	45	0.029
Cefotaxime	6	2	NS
Chloramphenicol	11	3	NS
Ciprofloxacin	11	4	NS
Gentamicin	13	5	NS
Kanamycin	10	3	NS
Nalidixic acid	46	18	NS
Nitrofurantoin	61	28	NS
Spectinomycin	31	14	NS
Streptomycin	142	66	NS
Sulfamethoxazole	123	55	NS
Tetracycline	136	62	NS
Trimethoprim	189	90	0.037
Trimethoprim/sulfamethoxazole	119	48	0.022
MDR	174	84	NS
NS, not significant.

Discussion

S. pullorum isolates showed an increasing resistance level from 1962 to 2010. In China, sulfamethoxazole, trimethoprim, trimethoprim/sulfamethoxazole, streptomycin and tetracycline were commonly used to control pullorum disease for a long time and this may explain the high resistance rates to these antibiotics (>50%). Salmonella serovars isolated from animals or foods in China also exhibited high resistance to the same antimicrobials (Chen et al., 2004; Yan et al., 2010; Yang et al., 2010; Lu et al., 2011). The decrease in resistant rates of some antimicrobials between 2000 and 2010 might be due to a ban or reducing use of specific antimicrobials from the Ministry of Agriculture of the People's Republic of China (MAPRC, 2002a, b).

S. Pullorum isolates developed an increasing tendency towards MDR under the selective pressure of antimicrobials. In the 1960s, the isolates had much lower MDRs (9.4%). MDR increased from 64.6 to 78.7% between 1970 and 1987. MDRs were at a high levels from 1990 (96.4%) to 2010 (96.7%). Studies have shown that the appearance of new antibiotic resistance mechanisms of Salmonella spp. of animal origin may directly influence the appearance of antibiotic-resistant human pathogens (Stobberingh & van den Bogaard, 2000). Veterinary clinicians should therefore use antimicrobials judiciously in order to prevent or delay the emergence of antibiotic-resistant S. Pullorum.

Integration was described as a hereditary unit composed of integrase coding genes, resistance gene cassettes and the integration site of the resistance gene cassettes in 1989 (Stokes & Hall, 1989). Integrons are the main pathway of genetic resistance for some bacteria. Resistance genes move among chromosomes, plasmids and transposons, leading to the spread of resistance among bacteria (Rowe-Magnus et al., 2002). There is a higher carrying rate of int1 than other types of integrons in Gram-negative bacteria, especially the Enterobacteriaceae (Gebreyes & Altier, 2002). Of all the S. Pullorum isolates tested, 22.3% carried int1 and all positives were isolated from 1990 to 2010.

The resistance rates of int1(+) isolates were higher than the negative isolates, but there were some differences among antimicrobials. Class 1 integrons are closely related to antimicrobial resistance and MDR in Enterobacteriaceae. There are a variety of gene cassettes in the variable region of int1 that encode resistance to sulfonamides and aminoglycosides (Rao et al., 2006). In the current study, we found a similar phenomenon of resistance rates to spectinomycin, streptomycin, sulfisoxazole and trimethoprim/sulfamethoxazole in int1(+) isolates. It seems likely that int1 plays an important role in MDR mechanisms of S. Pullorum infections in chickens.

Bacteria with biofilms may have an increased resistance to antimicrobials, ambient pressure and the host immune system (Arciola et al., 2001; Costerton et al., 2003; Szomolay et al., 2005). In order to understand the relationship between biofilms and antimicrobial resistance in S. Pullorum, we assayed the ability of the isolates to form biofilms. Our results showed differences among the isolates in that strains isolated from later years had higher rates of biofilm formation than the isolates from earlier years. The difference in resistance rates between the isolates with and without biofilm forming ability was not statistically significant except with regards to cefamandole, trimethoprim and trimethoprim/sulfamethoxazole. Bacteria within biofilms can significantly enhance antibiotic resistance relative to floating bacteria, but this is mainly manifested as the minimum bactericidal concentration, which may not be an indicator of in vivo antibiotic resistance of bacteria. A more accurate and objective evaluation method for correlating biofilms and antimicrobial resistance is therefore needed.

Acknowledgments

This project was supported by grants from the China National Science Foundation (No. 31101833), the Special Fund for Agroscientific Research in the Public Interest (No. 201303044), the Natural Science Foundation of Jiangsu Province, China (No. BK2011430) and the Sanxin Agricultural Programs of Jiangsu Province, China (No. SXGC[2012]383). The authors wish to express their thanks to Dr Howard Gelberg (Oregon State University) for manuscript editing.