Increasing Trend of Heterogeneous Vancomycin Intermediate Staphylococcus aureus in a Tertiary Care Center of Northern India

Abstract

Heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) strains are increasingly reported, and their association with vancomycin treatment failure is a well-known problem worldwide. A total of 500 clinical isolates of methicillin-resistant S. aureus were screened for hVISA by four different methods from May 2011 to May 2014. The number of strains screened for hVISA from May to April in 2011–2012, 2012–2013, and 2013–2014 were 198, 123, and 179, respectively. hVISA strains were studied by transmission electron microscopy (TEM) for cell wall thickness and also for their ability to form biofilm on a polystyrene microtiter plate. hVISA strains detected by four different methods—brain heart infusion agar with vancomycin with 4 mg/L/gradient plate/macro E-test/and glycopeptide resistance detection (GRD) E test—were as follows: 11.6%/10%/9%, and 9.5% in 2011–2012, 12.1%/9.7%/8.9%, and 10.5% in 2012–2013, and 13.9%/11.7%/11.1%, and 12.8% in 2013–2014, respectively. Population analysis profile–area under curve analysis confirmed hVISA in 4.5% (9/198), 6.5% (8/123), and 6.7% (12/179) in respective years; 24% (7/29) of hVISA isolates were nonsusceptible to daptomycin. TEM showed a significant increase in cell wall thickness of hVISA isolates (p<0.001) with a distinct reduction in their biofilm formation ability.

Introduction

Heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) has been detected worldwide.⁹ S. aureus with a reduced susceptibility to glycopeptides was first reported in 1997 in Japan and later, the strain was identified as hVISA.¹¹ Since then, hVISA strains have been reported throughout the world with prevalence ranging from 0% to 74%.^9,11,17 hVISA isolates are defined as strains of S. aureus with vancomycin minimum inhibitory concentration (MIC) within the susceptible range when tested by routine methods (previously a vancomycin broth MIC of ≤4 μg/ml and now a vancomycin broth MIC of ≤2 μg/ml) but where a proportion of the population of cells are in the vancomycin-intermediate range.^12,27 hVISA infections have been associated with vancomycin treatment failure and recurrence.¹ The reduced susceptibility to glycopeptides in vancomycin intermediate S. aureus (VISA) is due to the cell wall compositional changes that occur in the septum during cell wall synthesis in S. aureus during cell division.²¹

Vancomycin efficacy to treat methicillin-resistant S. aureus (MRSA) infection has been challenged due to different forms of reduced-susceptibility phenotypes (VISA and hVISA).³ Currently, several screening methods have been used to detect hVISA isolates¹¹ but screening and identification methods of hVISA are restricted to limited laboratories and are tediously time consuming.

Biofilm formation and its association with hVISA are contradictory.^13,29 VISA strains were associated with biofilm formation on biomedical devices and presumed to be a significant initial step in the pathway to vancomycin resistance.²⁵ An association of biofilm formation with hVISA strains from hospitals in India has not been reported. Reports on prevalence and characterization of hVISA in hospitals from different countries, including India, are limited. This study aimed at identifying hVISA strains in clinical isolates of MRSA by various methods, their cell wall thickness and ability to form biofilm.

Materials and Methods

Bacterial strains

A total of 500 MRSA isolates recovered from various clinical specimens of Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS, Lucknow, India), a 900-bed tertiary care referral hospital, were included in the study. To prevent reversion of resistance, freshly isolated strains were used in this study. Only one isolate per patient was included. All the isolates were stored in cryo-vials at −80°C until MIC testing was performed. Mu3 (hVISA), Mu50 (VISA), and a vancomycin-sensitive S. aureus (VSSA) strain ATCC 29213 were used as reference organisms for the antimicrobial susceptibility testing and population analysis profile–area under curve (PAP-AUC) method.

Antimicrobial susceptibility testing

MICs were performed by standard E-test (bioMerieux, Marcyl’ Etoile, France) procedure. Brain heart infusion agar (BHIA; Oxoid Ltd., Basingstoke, England) plates were swabbed with 0.5 McFarland standard suspensions of test organisms and allowed to dry. Vancomycin, teicoplanin, and daptomycin E-strips were placed and the plates were incubated at 37°C for 18 hr.

Screening for hVISA

MRSA strains were screened for hVISA by four different methods—BHIA plates containing 4 mg/L vancomycin (BHIV4), gradient plates, macro E-test (MET), and E-test glycopeptide resistance detection (GRD).^11,17,31 Ten microliter of 0.5 McFarland bacterial broth suspensions were inoculated on to BHIV4. Growth at 24 hr denoted VISA and at 48 hr, it denoted hVISA. Strains were tested in batches of 10; strains Mu3 and Mu50 were used as positive controls; and S. aureus ATCC 29213 was used as a negative control with each batch. The pure vancomycin powder was obtained from Sigma Chemical Co. (St. Louis, MO).

Gradient plates were made by allowing 25 ml of BHIA containing 4 mg/L of vancomycin to be set at a 12° angle in a 10 cm square Petri dishes. This slope was subsequently overlaid with 25 ml of BHIA and left to set horizontally. After 24 hr of incubation in brain heart infusion broth (BHIB), each culture was adjusted to turbidity equal to McFarland 0.5 and a standard loop full (10 μl) was streaked across the vancomycin gradient plate (0–4 mg/L). Ten clinical isolates plus Mu3 in duplicate as a positive control (the repetition of Mu3 providing reproducible data) were tested on each plate. The growth along the vancomycin gradient was measured after 48 hr. The gradient plates were analyzed using ratio of the growth distance between MRSA and Mu3 on the same plate. The ratio of ≥1 denoted a hVISA phenotype.

MET was performed as follows: 2.0 McFarland suspensions of the test isolates in saline were swabbed on to the BHIA plates and allowed to dry; vancomycin and teicoplanin E-test strips were applied. After incubation for 48 hr at 37°C, the MICs were noted. The criterion applied to detect hVISA by the MET was the appearance of one or more subpopulation colonies with MIC≥4 mg/L.

E-test GRD (bioMerieux) was performed according to the manufacturer's instructions. Mueller-Hinton agar containing 5% sheep blood was inoculated with a 0.5 McFarland suspension of overnight growth followed by GRD strip placement. After 24 and 48 hr of incubation in ambient air at 35°C, GRD tests were read. A positive GRD result for hVISA was a vancomycin or teicoplanin MIC of ≥8 μg/ml at 24 or 48 hr.

Isolates that displayed hVISA profile by any of the methods mentioned earlier were further analyzed using the modified PAP-AUC method as previously described.³¹ Briefly, vancomycin PAP-AUC was performed by serial dilution of an overnight (BHIB; Hi-media, Mumbai, India) culture and inoculation on BHIA containing 0–8 mg/L vancomycin. Colonies were counted after incubation for 48 hr at 35°C, and the number of CFUs/ml versus vancomycin concentration was plotted on a graph. The hVISA strain Mu3 and the vancomycin-susceptible S. aureus strain ATCC 25923 were tested in parallel as positive and negative controls, respectively. An isolate was defined as hVISA if the PAP-AUC of the test strain to Mu3 was ≥0.9.³¹

Assessment of biofilm formation

The biofilm formation ability of hVISA strains on a polystyrene microtiter plate was assessed as previously described, with few modifications.¹⁰ Ten clinical isolates of hVISA and VSSA were cultured onto blood agar and incubated aerobically at 35°C for 24 hr. The grown cultures were used for preparation of bacterial suspensions in sterile distilled water with densities adjusted to 0.5 McFarland standards. The wells of a sterile 96-well flat-bottomed plastic tissue culture plate with a lid were filled with 180 μl of BHIB. Aliquots of 20 μl of bacterial suspensions were added into each well. Isolates were tested in triplicate, at least twice. The plates were incubated aerobically for 24 hr at 35°C; the content of each well was then removed, and the wells were washed four times with 200 μl of sterile distilled water. The plates were emptied, allowed to air dry at room temperature for 15 min, and stained with 200 μl per well of 0.1% crystal violet for 10 min. After being dried, the crystal violet was solubilized by adding 200 μl of 95% ethanol and the absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay reader (Spectra easy microplate reader; Inverness medicals, New Delhi, India). Each assay was performed thrice.

Electron microscopy

hVISA strains were sub-cultured on Columbia blood agar plates and grown at 37°C for 24 hr. Each strain was suspended in phosphate-buffered saline, pelleted by centrifugation, and re-suspended in fixative as previously described.⁶ Briefly, ultrathin sections (50–70 nm) were cut with an ultra-microtome, stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (FEI Technai G2 Sprint TWIN, Hillsboro, OR).

Statistical analysis

The statistical significance of the data was evaluated by Student's t-test, and the PAP-AUC curve was drawn and analyzed using Graph Pad Prism 5.0 (Graph Pad Software, Inc., San Diego, CA).

Results

A total of 500 MRSA isolates were tested for hVISA prevalence over a 3 year period (May 2011–May 2014). Distributions of MRSA isolates from different specimens were 241, 101, 137, and 21 from pus (obtained from skin and skin structure infection, and deep-seeded abscess), blood, sputum, and urine, respectively. Number of strains screened for hVISA from May to April in 2011–2012, 2012–2013, and 2013–2014 were 198, 123, and 179, respectively. Strains identified hVISA by four different screening methods—BHIV4/gradient plate/MET/and GRD E-test was as follows: 11.6%/10%/9%, and 9.5% in 2011–2012, 12.1%/9.7%/8.9%, and 10.5% in 2012–2013, and 13.9%/11.7%/11.1%, and 12.8% in 2013–2014, respectively. PAP-AUC analysis confirmed hVISA in 4.5% (9/198), 6.5% (8/123), and 6.7% (12/179) in respective years with an overall prevalence of 5.8% (29/500). Prevalence of hVISA increased from 4.5% in 2011–2012 to 6.5% in 2012–2013 and to 6.7% in 2013–2014 (Fig. 1). Positive predictive values (PPVs) of BHIV4, gradient plate, MET, and GRD E-test methods were 46.2% (29/63), 54.7% (29/53), 59.1% (29/49), and 52% (29/55), respectively. Isolation rates of hVISA from different clinical specimens were as follows: 19.1% (4/21) from urine, 12.8% (13/101) from blood, 4.1% (10/241) from pus, and 1.45% (2/137) from sputum. Twenty (69%) and 25 (86.2%) of 29 hVISA isolates had a vancomycin MIC range from 1.5 to 2.0 mg/L and a teicoplanin MIC range from 3 to 8 mg/L, respectively (Figs. 2 and 3); 24% (7/29) of hVISA isolates were nonsusceptible to daptomycin. Glycopeptides and daptomycin MIC values with their PAP-AUC ratios of all the hVISA isolates are shown in Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr. In addition, 10 (2%) among 500 MRSA isolates had vancomycin MIC between 4 and 8 mg/L and these isolates were categorized as VISA.

FIG. 1.

Year-wise distribution of hVISA by four different screening methods (MET, BHIV4, GRD E-Test, GPM) and PAP-AUC confirmatory method. BHIV4, brain heart infusion agar with vancomycin 4 mg/L; GPM, gradient plate with vancomycin gradient from 0 to 4 mg/L; GRD E-Test, glycopeptide resistance detection Epsilometer test; hVISA, heterogeneous vancomycin intermediate Staphylococcus aureus; MET, macro E-test; PAP-AUC, population analysis profile–area under curve.

FIG. 2.

Minimum inhibitory concentrations (MICs) of hVISA isolates detected by four different screening methods (BHIV4, MET, GPM, GRD E-test) and PAP-AUC (gold standard for hVISA detection) to vancomycin.

FIG. 3.

Minimum inhibitory concentrations of hVISA isolates detected by four different screening methods (BHIV4, MET, GPM, GRD E-test) and PAP-AUC (gold standard for hVISA detection) to teicoplanin.

The TEM results showed significantly (p≤0.001) higher cell wall thickness in all 29 clinical isolates of hVISA (n=20: 48.82±3.2 nm; n=5: 24.71±2.05 nm and n=4: 44.18±1.85 nm) compared with the VSSA ATCC 29213 strain (n=1: 18.16±2.6 nm). Similarly, a much thicker cell wall was also observed in positive controls of hVISA (Mu3) and VISA (Mu50)—34.59±2.8 and 51.32±51.32 nm, respectively.

Analysis of biofilm formation ability of hVISA showed a significant (p=0.0019) reduction in biofilm production when compared with VSSA strains as reflected by mean optical density values (ODs) of VSSA 1.96 (range, 0.8–2.9) and hVISA 0.87 (range, 0.6–1.2). The mean OD of strain ATCC 29213 (glycopeptide sensitive strain) was 2.2 and of Mu3 strain (hVISA control strain) was 1.19 (Fig. 4).

FIG. 4.

Quantification of biofilm formation was determined by crystal violet staining, and the absorbance at 570 nm was read using an enzyme-linked immunosorbent assay plate reader.

Discussion

Screening for hVISA is not routinely performed in clinical laboratories of India. Therefore, the true prevalence of hVISA remains largely unknown from this geographic region, whereas inappropriate use of antibiotics and bacterial disease burden is quite high in this region.^8,20

In this study, four different screening methods (BHIV4, gradient plate, MET, and GRD E-test) were used to detect year-wise prevalence of hVISA among the clinical isolates of MRSA and compared the PPVs of these methods. We observed that PPV of MET was the highest (59.1%) followed by gradient plate (54.7%), E-test GRD (52.7%), and BHIV4 (46%) methods. Our result suggests that MET may be used as a tool for rapid screening of hVISA.

In an earlier study from Hong Kong, 40.3% (21/52) MRSA strains were positive for hVISA by the initial BHIAV4 screening method but only 5.7% (3/52) strains confirmed hVISA by PAP.³⁰ In this study, we noted that 12.6% (63/500) strains were initially positive for hVISA by any of the four screening methods but PAP confirmed hVISA only in 5.8% (29/500) strains. The difference in screen positive between the two studies might be related to differences in sample size, geographical locations, and type of health-care settings.

A review summarized that the E-test performed with a 2.0 McFarland standard on BHIA gave the most precise results, with high sensitivity (93%) and specificity (97%).¹⁸ This study also showed that MET had the highest PPV (59%) among the four screening methods followed by E-test GRD with PPV 54.7%. Previously, Richter et al. observed that the PPV of E-test GRD was only 16.2% for hVISA screening from 43 different medical centers that included 4,210 S. aureus strains.²³ The reason for higher PPV of E-test GRD in our study might be due to selective screening of MRSA isolates. Wide variability with large numbers of false positive and negative results for the gradient plate method had been reported.³¹ Moreover, preparation of the gradient plate is very tricky and cumbersome, making it difficult to be used in routine diagnostic laboratories for hVISA detection.

In 2004, a study reported the prevalence of hVISA among MRSA isolates from 12 Asian countries; the prevalence varied from 0% to 8.2%, with 6.3% being reported from South India.²⁷ In this study, prevalence of hVISA was similar to an earlier study from the southern part of the country but we observed an increasing trend over the consecutive 3 years from 4.5% in 2011–2012 to 6.5% in 2012–2013 and 6.7% in 2013–2014. Several research groups had reported the prevalence of hVISA that ranged from 0% to 74% from the different parts of the world.⁷ One study by Ariza et al. reported hVISA in 14 (74%) of 19 isolates.² This high isolation of hVISA might be related to small sample size, methodology used, and selection bias, that is, only orthopedic surgery patients with MRSA infections were included. We observed ∼1.5-fold increase in hVISA prevalence over the study period. Rybak et al. reported ∼6.1-fold increased hVISA prevalence in Detroit, Michigan over a 22 year period.²⁴ One limitation of our study is that we did the PAP-AUC to only screen positive hVISA isolates, hence it is possible that few hVISA strains might have not been detected. However, one distinct feature of our study is that we used all the four available hVISA screening methods so that hVISA isolates were not missed out. Moreover, the PAP-AUC method is cumbersome and labor intensive for testing all the strains. Hence, it is routine practice that the strains that are positive by screening method(s) are usually subjected to PAP-AUC analysis.

Recognition of vancomycin-resistant S. aureus (VRSA), VISA, and hVISA has caused a great deal of concern, as they have been associated with clinical failures.^15,22 VRSA and VISA strains are easy to identify by determining their MIC, but it is difficult to detect hVISA strains since their MIC values are within the susceptibility range. In this study, 20 (68.9%) and 25 (86.2%) of 29 hVISA strains had vancomycin MIC between 1.5 and 2 mg/L and teicoplanin between 3 and 8 mg/L, respectively. Over the past few years, uneven patterns of vancomycin MICs within the S. aureus populations have been the major consideration. Previous studies reported poorer outcomes for vancomycin treatment of MRSA infections with higher vancomycin MICs but still within the range of susceptibility.^26,28 Moreover, in this study, 10 (2%) of 500 MRSA isolates were VISA (vancomycin MIC between 4 and 8 mg/L).

Daptomycin is considered an alternative treatment modality for VRSA, VISA, and hVISA except respiratory infections. In this study, 24% (7/29) of hVISA isolates were found nonsusceptible to daptomycin. Similar to our study, one Australian study also reported 26% (7/26) of hVISA isolates that were nonsusceptible to daptomycin by E-test.²⁶ Kelley et al. reported that 3% of MRSA isolates with vancomycin MIC>2 were nonsusceptible to daptomycin.¹⁶ Previous in vitro studies suggest that daptomycin may have a lower rate of in vitro killing against hVISA isolates.⁴ An association between cell wall thickening and reduced daptomycin susceptibility in glycopeptides intermediate S. aureus isolates had been observed.⁵ Whether the reduced daptomycin susceptibility in some hVISA strains can be attributed to cell wall thickening or to other mechanisms remains to be established. To the best of our knowledge, this report describes the first report of daptomycin nonsusceptible hVISA isolates from India.

Cell wall thickness is a consistent feature of hVISA isolates.¹² Results of electron microscopy in this study also showed a significant increased (1.3–2.6-folds) cell wall thickness in clinical hVISA isolates compared with VSSA isolates. Although the exact mechanism of thickening of the cell wall in hVISA has not been determined, it is believed that the thickened cell wall prevents the diffusion of vancomycin into the cytoplasmic membrane, leading to treatment failure.

Biofilm formation may play an important role in pathogenesis of VISA or in hVISA infections associated with prosthetic materials.¹⁴ Increased biofilm production by laboratory-induced hVISA compared with the parent strain was reported.²⁵ On the contrary, Howden et al. observed a noteworthy reduction in biofilm formation by clinical hVISA strains compared with VSSA parent strains.¹³ In this study, we also observed significant reduction in biofilm production by hVISA strains compared with VSSA. The exact reason for differences in biofilm formation is difficult to explain; however, an earlier study correlated strong biofilm formation with loss of agr function,¹³ while another study correlated it with agr type II.¹⁹ Further studies are required to understand the differences in biofilm formation by hVISA isolates in different studies.

In conclusion, although the prevalence of hVISA was low, an increasing trend over the periods was observed at our center. Detection of daptomycin nonsusceptible hVISA isolates is a major concern, and, thus, it calls for judicious use of this drug. Since the confirmatory PAP-AUC method is cumbersome and labor intensive, each clinical laboratory should use at least one screening method for early detection of hVISA. Further studies are needed to provide a better insight into the development of daptomycin nonsusceptibility in some hVISA strains.

Footnotes

Acknowledgments

The authors are thankful to Prof. Keiichi Hiramatsu (Tokyo, Japan) for providing the strains of Mu3 (hVISA) and Mu50 (VISA). A.S. acknowledges the financial assistance received from the Indian Council of Medical Research, Government of India, through senior research fellowship grant no. 80/675/10-ECD-I. S.K.S. acknowledges the support received from the Council of Scientific and Industrial Research (CSIR), Government of India, through junior research fellowship grant no. (CSIR-JRF) 09/590(0147)/2010-EMR-1. Financial assistance received for the study through SGPGIMS intramural grant no. 2013-30-IMP-EXP/177 is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

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