Evaluate the frequency distribution of nonadhesive virulence factors in carbapenemase-producing Acinetobacter baumannii isolated from clinical samples in Kermanshah.

BACKGROUND: Acinetobacter baumannii which is a Gram-negative bacterium can cause several different infections. The appearance of carbapenemase-producing A. baumannii in recent years has made the treatment process more difficult. The identification of virulence factors (VFs), such as nonadhesives in A. baumannii, helps to fight against related infections.
MATERIALS AND METHODS: A total of 104 samples from teaching hospitals in Kermanshah, Iran, were collected during a 24 months period (2011-2013). Sample identification was first carried out by biochemical tests, and then their susceptibility to carbapenems was determined using the Kirby-Bauer method. For confirmation of carbapenemase-producing A. baumannii, polymerase chain reaction (PCR) was done for carbapenemase-encoding genes. In addition, the frequency of nonadhesive VFs in carbapenemase-producing isolates was determined by PCR.
RESULTS: There were 50 isolates that were identified as carbapenemase-producing A. baumannii. The PCR results showed; 40 isolates (80%) for traT, 17 isolates (34%) for cvaC, and 8 isolates (16%) for iutA, and these encode serum resistance, colicin V and aerobactin, respectively. No significant correlation was observed between these three genes.
CONCLUSIONS: The mechanism of A. baumannii virulence has always been in question. The role of VFs has also been recognized in other Gram-negative bacteria. According to the prevalence of traT, cvaC and iutA, as nonadhesive VFs, we can suggest that they could be the main mechanism of carbapenemase-producing A. baumannii pathogenesis.

INTRODUCTION

Acinetobacter baumannii is an aerobic, nonfermentative Gram negative Coccobacillus that can survive for prolonged periods in the environment and can cause nosocomial infections, (urinary tract and wound infections).[123] Capability to live under critical circumstances makes it a major pathogen in hospitals.[4] Extensive use of antimicrobial agents in clinical cases has contributed to the appearance of resistant A. baumannii strains.[5] The antimicrobial agent recommended against A. baumannii infections are usually carbapenems. Increasing resistance to carbapenems in A. baumannii infections in the past decade[1] is creating therapeutic challenges.[6] One of the characteristics of pathogenic bacteria is that they contain virulence factors (VFs).[7] The bacterial VFs include adhesive and nonadhesive types. Adhesive VFs can attach by methods such as fimbriae. Nonadhesive VFs include: Diverse siderophores, such as; yersiniabactin (fyuA), aerobactin (iutA), invasins (IbeA), serum resistance (traT), and colicin V production (cvaC).[8] The purposes of this work was to detect the genes codifying nonadhesive VFs, and to evaluate the frequency distribution of nonadhesive VFs in carbapenemase-producing A. baumannii isolated from clinical samples.

MATERIALS AND METHODS

Bacterial collection

One-hundred four samples were isolated from patients hospitalized in three hospitals affiliated with Kermanshah University of Medical Sciences, Iran during March 2011-March 2013.

Identification and antimicrobial susceptibility testing

The strains were identified as A. baumannii by conventional biochemical tests and API 20NE kit (version 6.0, bio-Mérieux, France).[9] Susceptibility to carbapenem was performed according to the standard Clinical and Laboratory Standards Institute guidelines to check their resistance to carbapenems using imipenem (10 μg) and meropenem (10 μg) (MAST, Merseyside, UK).[10] Carbapenem-resistant isolates were chosen for identifying nonadhesive VFs frequency. Escherichia coli MK1 and MK2 were used for ibeA and fyuA as a positive control. For VF traT, cvaC and iutA one amplified a polymerase chain reaction (PCR) product of each gene sent to Sinaclon Company for sequencing and the results were used as the positive control after nBlast alignment.

Polymerase chain reaction

Bacterial DNA was released from whole microorganisms by boiling. Bacteria were harvested from 1 ml of an overnight broth culture, suspended in 200 μl of sterile water, and incubated at 100°C for 10 min. Following centrifugation of the lysate, a 150 μl sample of the supernatant was stored at −20°C as a template DNA stock.[11] The primers used in this study are shown in Table 1.[8] Each reaction mixture (15 μl) contained 2 μl of DNA, 0.5 mM of each primer, 1 U of Taq DNA polymerase (Sinaclon), the four deoxynucleoside triphosphates (each at 200 μM) and 1.5 mM MgCl2. PCR was performed under predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, at each specific annealing temperature [Table 1] for 30 s, and 72°C for 45 s ending with a final extension step at 72°C for 5 min. The PCR product was run and visualized on 1.5% agarose gels then were stained with ethidium bromide. PCR screening was done for the carbapenemase-encoding genes including blaOXA-23-like and blaOXA-24-like. PCR analysis was performed using the primers described previously.[12]

Table 1

Primers to VFs used in the polymerase chain reaction

Statistical analysis

A statistical comparison of the frequencies of nonadhesive VFs in carbapenemase-producing A. baumannii isolates was conducted by Chi-squared test (variables were analyzed by Chi-square test). P = 0.05 was considered as statistically significant.

RESULTS

One-hundred four A. baumannii specimens were collected from three hospitals in Kermanshah (Iran). Fifty isolates were resistant to imipenem and meropenem and also positive for one of the carbapenemase-encoding genes (blaOXA-23-like, blaOXA-24-like); these isolates were detected as carbapenemase-producing isolates for future analysis. The carbapenemase resistant bacteria were isolated from 35 (70%) males to 15 (30%) females, and the distributions of carbapenemase-producing A. baumannii, according to age, are shown in Figure 1. Nonadhesive VFs were analyzed by a PCR assay. Products of the PCR assay are shown in Figure 2. Frequencies of nonadhesive VFs include: Serum resistance (traT), colicin V (cvaC), and aerobactin (iutA), were; 40 isolates (80%), 17 isolates (34%), and 8 isolates (16%), respectively. However, the genes fyuA, ibeA, and pathogenicity-associated islands (PAI), codifying for; yersiniabactin, invasion and PAIs, respectively, were not detected by means of a PCR with specific primers. There were 4 (8%) isolates that carried all three genes. However, no VF was found in 5 (10%) isolates. Eight (16%) isolates carried both traT and cvaC (P = 0.27), 6 (12%) isolates carried traT and iutA (P = 0.18), and 4 (8%) isolates carried cvaC and iutA (P = 0.41). There were no significant correlations found between these three genes. Distribution of traT, cvaC and iutA did not correlate with gender [Table 2].

Figure 1

Distribution of carbapenemase-producing Acinetobacter baumannii according to the age of patients

Figure 2

Patterns of agarose gel electrophoresis showing polymerase chain reaction amplification products for the nonadhesive virulence factors genes. Lane M, DNA molecular size marker (100 bp ladder; Sinaclon). (a) Lane 1: traT (290 bp); Lane 3: cvaC (680 bp); Lane 5: iutA (300 bp); Lane 2, 4, 6: Negative control. (b) Lane 2: fyuA (785 bp); Lane 4: ibeA (170 bp); Lane 3 and 5: Negative control

Table 2

Distribution of traT, cvaC and iutA according to sex

DISCUSSION

Treatment of infections with opportunistic pathogens such as A. baumannii has been a great challenge. Carbapenems are the first-line of treatment of Acinetobacter infections, but nowadays resistance to them is reported.[8] In this study, 50 isolates (48%) were recognized as carbapenemase-producing phenotypes out of 104 clinical isolates of A. baumannii. 25-50% incidence of resistance to carbapenems is previously reported in Iran.[1314] Park et al. reported resistance to imipenem and meropenem, 31.7% and 34.9% respectively from 63 isolates of Acinetobacter in South Korea.[15] Resistant rate to carbapenem in A. baumannii isolates investigated in this study is different compared to other studies, which may be due to geographical differences in isolates.

Fifty carbapenemase-producing isolates were examined in terms of having six nonadhesive VFs including traT (serum resistance), cvaC (colicin V), iutA (aerobactin), ibeA, (invasion), fyuA (yersiniabactin) and PAI. Frequency of traT, cvaC and iutA factors were 40 (80%), 17 (34%) and 8 (16%) isolates, respectively. VFs cause infection in most Gram negative pathogenic bacteria. Of these, nonadhesive VFs play a key role in bacterial survival in special conditions such as in human blood and exposure to serum and poor iron environments that cause diseases.[8] Compared to other Gram-negative pathogens, relatively few VFs have been identified for A. baumannii.[16] Serum resistance (traT) is one of the nonadhesive factors investigated in this study. Studies indicate that serum resistance is the ability of the bacterial cell to resist the lytic effects of serum and to invade and survive in the human blood stream. Doughari et al. (2012) studied 14 samples of Acinetobacter and indicated that 7 isolates (50%) had serum resistance phenotypically.[17] King et al. in the USA found that from 7 A. baumannii isolates, 3 isolates (43%) had serum resistance phenotypically.[18] The results obtained from carbapenemase-producing A. baumannii isolates in this study show that genotypic frequency of traT gene is higher than in other studies. Of course, this difference could be due to phenotypic and genotypic detection techniques for these VFs. There is also a direct relationship between serum resistance and biofilm formation, and hence they increase bacterial pathogenicity.[18] As a result, considering the existence of 80% traT gene in this study, more pathogenicity is expected for these isolates. Another VF described in A. baumannii is the production of siderophores by isolates growing under iron limiting conditions. These bacteria are capable to express high-affinity iron uptake systems composed by siderophores. In Braun study, all A. baumannii strains were able to grow under iron limiting conditions, and they concluded that this bacterium produces siderophores.[8] Unlike our study, the genes iutA and fyuA from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the Braun studied isolates. Considering the frequency of iutA gene in this study,[17] it seems that iutA siderophore unlike fyuA in Acinetobacters isolated from sputum/tracheal secretion, is more important VF compared to urinary isolates. Another gene that was positive in this study is cvaC, which was observed in 34% of cases. cvaC, which encodes colicin V, have been proposed to confer enhanced virulence through their carriage of other specific VFs, including the aerobactin system and serum survival genes, such as traT.[19]

CONCLUSION

Carbapenemase-producing A. baumannii strains are among the most important agents of nosocomial infections in our hospital and worldwide. Three VFs of traT, cvaC and iutA with frequency of 80%, 34%, and 16%, respectively, revealing a role in the pathogenesis of these isolates and also may be have the most impact on existence for long time by carbapenemase-producing A. baumannii in environment of hospital. Frequent screening for these VFs may contribute to better infection control measures and to the investigate new treatment options.

Financial support and sponsorship

Kermanshah University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.